Guayule with increased rubber production and yield

ABSTRACT

A reduction in the amount of functional PaAos in guayule results in the production of increased amounts rubber compared to the amount of rubber produced by wild-type guayule having a non-reduced amount of functional PaAos. Further, the guayule with reduced amount of functional PaAos are larger than wild-type guayule and thus have larger rubber yield per acre than wild-type guayule. Reduction of the amount of functional PaAos in guayule can be caused by genetic alterations in PaAos. Guayule having PaAos with a specific amino acid sequence produces more rubber than guayule with PaAos having a different amino acid sequence. Thus, one can use the sequence differences as a biomarker for selecting high rubber producing guayule plants.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Patent Application62/504,762 filed on May 11, 2017, contents of which are expresslyincorporated by reference herein.

BACKGROUND OF THE INVENTION Sequence Listing

The Sequence Listing submitted via EFS-Web as ASCII compliant text fileformat (.txt) filed on May 10, 2018, named “SequenceListing_ST25”,(created on May 9, 2018, 24 KB), is incorporated herein by reference.This Sequence Listing serves as paper copy of the Sequence Listingrequired by 37 C.F.R. § 1.821(c) and the Sequence Listing incomputer-readable form (CRF) required by 37 C.F.R. § 1.821(e). Astatement under 37 C.F.R. § 1.821(f) is not necessary.

Field of the Invention

This invention relates to altered guayule plants that grow larger andproduce more rubber than non-altered guayule plants, when grown underthe same conditions. The altered guayule contain the cDNA sequence ofParthenium argentatum Allene oxide synthase (PaAos) in the reversecomplement orientation under control of a heterologous promoter whichreduces the production of PaAos via RNAi. Other types of geneticalternations can be made in guayule to reduce the functionality ofPaAos. Kits for identifying such altered guayule, methods for identifythe altered guayule, and methods of increasing rubber yield in guayulevia reducing PaAos translation are also included.

Description of Related Art

Natural rubber is synthesized by more than 2,500 plant species (Cornish,et al., J. Nat. Rubber Research 8:275-285 (1993); Cornish, K.,Phytochemistry 57:1123-1134 (2001)). Rubber is produced by these plantsas a secondary metabolite with no clear indication of its function inplant cells. Possible reasons on why these species synthesize rubber areto defend themselves against pathogens and insect attacks, repair tissuedamages caused by mechanical wounding and protect cell damage induced byenvironmental stresses (Demel, et al., Biochim. Biophys. Acta.1375:36-42 (1998); Tangpakdee and Tanaka, J. Rubber Res. 1:14 (1998);Vereyken, et al., Biochim. Biophys. Acta, 1510:307-320 (2001); Kim, etal., Plant Cell Physiol., 412-414 (2003) and references therein; Konno,K., Phytochemistry, 1510-1530 (2011); and Sarkar, J., Rubber Science,228-237 (2013)). According to a 2014 market report, the rubber thatthese plants produce accounted for $16.5 billion in trade worldwide(rubberworld.com/RWmarket_report.asp). Even more so, the end productsmade from natural rubber, including tires for the transportationindustry, sports equipment, medical devices, and more, are indispensablein our everyday life. The Hevea tree is the main source of naturalrubber but concerns exist as it is limited geographically to tropicalclimates, mainly in Southeast Asia, is susceptible to diseases, andproduces rubber that causes allergic reactions. Clearly, an alternativesource for the production of natural rubber is very important to reduceeconomic risk and safeguard human health.

One plant known to be a promising source of natural rubber is guayule(Parthenium argentatum, Gray), a desert shrub native to the southwesternUnited States and northern Mexico (Mooibroek and Cornish, Appl.Microbio. and Biochem. 53:355-365 (2000); van Beilen and Poirier,Critical Reviews Biotech. 27:217-231 (2007)). The majority of rubbersynthesis in guayule occurs during the cold season. Guayule synthesizesrubber within subcellular organelles called rubber particles (Archer andAudley, Bot. J. Linnean Soc. 94:181-196 (1987)) stored in the parenchymacells of stembark tissues (Gilliland, M. v., Protoplasma, 169-177(1984)); Macrae, S. G., Plant Physiol., 1027-1032 (1986)). Naturalrubber synthesis is initiated by the action of allylic pyrophosphatesinitiators (Cornish and Siler, J. Plant Physiol., 301-305 (1995)),usually farnesyl pyrophosphate (FPP). Then, the monomerisopentenyl-pyrophosphate (IPP), produced by the mevalonic acid pathway(MEV) in the cytosol and the methylerythritol phosphate (MEP) pathway inthe plastid (Mooibroek and Cornish (2000); van Beilen and Poirier,TRENDS in Biotech., 522-529 (2007)) elongates the rubber chain. Rubbersynthesis is mediated by rubber transferases requiring magnesium ions ascofactor (Da Costa, et al., Phytochemistry 67(15): 1621-1628 (2006)).

The proposed model for the structure of rubber particles consists mostlyof hydrophobic cis-polyisoprene units (natural rubber) encapsulatedinside a protein and phospholipid surface monolayer (Nawamawat, et al.,Colloids and Surfaces A: Physicochemical and Engineering Aspects,390:157-166 (2011); Sansatsadeekul, et al., J. Biosci. and Bioeng,111:628-634 (2011)). The phospholipids serve to stabilize and solubilizethe otherwise insoluble (rubber) product. Guayule rubber particlesinclude several proteins (Whalen, et al., Development of crops toproduce industrially useful natural rubber. Chapter 23 in IsoprenoidSynthesis in Plants and Microorganisms: New Concepts and ExperimentalApproaches, Bach and Rohmer (eds.), DOI 10.1007/978-1-4614-4063-5_23,Springer Science+Business Media NY, 329-345 (2013)) of which Aos hasbeen found to be the most abundant (Backhaus, et al., Phytochemistry30:2493-2497 (1991)). Aos is well-known as an enzyme in the jasmonicacid biosynthetic pathway (Harms, et al., Plant Cell, 1645-1654 (1995);Wang, et al., Plant Mol. Biol., 783-793 (1999); Schaller, F., J. Exper.Botany, 11-23 (2001)). The role of Aos in rubber biosynthesis, and thereason for the abundance of Aos protein on guayule rubber particlesurfaces, is not known (Whalen, et al. (2013)).

The need exists for a method to increase rubber production in alteredguayule compared to rubber production amounts in non-altered guayule inorder to improve the commercial attractiveness of using guayule rubberas a replacement of synthetic rubber and Hevea rubber. Further, a needexists for increasing the rubber yield per acre obtained from alteredguayule compared to the rubber yield per acre obtained from non-alteredguayule. This greater rubber yield results from the altered guayulebeing larger in size than non-altered guayule of similar age. A needalso exists for altered guayule that produce more rubber than the amountof rubber produced by non-altered guayule. A need also exists foraltered guayule that have a larger size than similarly aged non-alteredguayule because the altered guayule that are larger than the non-alteredguayule will possess more tissue for storage of rubber and thus generategreater rubber yield per acre than the rubber yield per acre ofwild-type guayule. And a need exists for biomarkers which distinguishbetween low rubber producing and high rubber producing guayules.

BRIEF SUMMARY OF THE INVENTION

It is an object of this invention to have altered guayule, parts ofaltered guayule, and progeny of the altered guayule, the altered guayulecontaining a mutation that causes the altered guayule to produce morerubber than the amount of rubber produced by a non-altered guayule. Itis a further object of this invention that the mutation in the alteredguayule can be one or more of (i) an alteration in a Partheniumargentatum Allene oxide synthase (PaAos) codon encoding an amino acid toa stop codon, (ii) an alteration of PaAos' translation initiation codonto another codon, (iii) an alteration in PaAos ribosome binding site'ssequence, (iv) an alteration in one or more PaAos splice site codons,(v) a deletion of part or all of PaAos' sequence, (vi) an insertion ofDNA into PaAos, and (vii) an alteration in one or more PaAos DNA codonsequences to encode a non-conservative amino acid. It is an object ofthis invention that each of these mutations reduces the altered PaAos'functionality compared to the amount of PaAos functionality in anon-altered guayule and that the reduced PaAos functionality causes thealtered guayule to produce an increased amount of rubber compared to theamount of rubber produced by the non-altered guayule. It is anotherobject of this invention that the alteration of one or more PaAos DNAcodon sequences to encode a non-conservative amino acid occurs at aminoacids located at 318, 332, 336, 339, 359, 408, 411, and 459 with PaAos'sequence (see SEQ ID NO: 10, 13, and 15). It is a further object of thisinvention that the amino acids being changed to non-conservative aminoacids are D318, S332, E336, R339, S359, I408, S411, and/or L459. Anotherobject of this invention is that the non-conservative amino acidsubstitutions are not N318, V408 and/or W459. It is another object ofthis invention to have an altered cell, germplasm, and an altered seedof the altered guayule, each containing the mutation.

It is an object of this invention to have a method of producing analtered guayule that contains a mutated PaAos and produces more rubbercompared to the amount of rubber produced by a non-altered guayule. Itis another object of the invention that the method involves exposing anon-altered guayule cell or seed to a mutagen to produce a mutatedguayule cell or seed with the mutated PaAos, selecting one or more ofthe mutated guayule cells or seeds containing the mutated PaAos whichencodes an altered PaAos with reduced functionality compared to anon-altered PaAos's functionality, and growing the selected mutatedguayule cell or seed containing the mutated PaAos to produce an alteredguayule that produces the altered PaAos with reduced functionality andan increased amount of rubber compared to the amount of rubber producedby the non-altered guayule. It is another object of this invention thatthe mutated PaAos contains at least one of (i) an alteration of a PaAoscodon encoding an amino acid to a stop codon, (ii) an alteration ofPaAos' translation initiation codon to another codon, (iii) analteration of PaAos ribosome binding site's sequence, (iv) an alterationof one or more PaAos splice site codons, (v) a deletion of part or allof PaAos' sequence, (vi) an insertion of DNA into PaAos, and (vii) analteration of one or more PaAos codon sequences to encode anon-conservative amino acid. It is another object of this invention thatthe alteration of one or more PaAos codon sequences to encode anon-conservative amino acid occurs at amino acids located at 318, 332,336, 339, 359, 408, 411, and 459 with PaAos' sequence (see SEQ ID NO:10, 13, and 15). Another object of this invention is that thenon-conservative amino acid substitutions are not N318, V408 and/orW459. It is another object of this invention to have an altered cell,germplasm, and an altered seed of the altered guayule produced by thismethod and each containing the mutation.

It is an object of this invention to have a method for producing apopulation of high rubber producing guayule plants or seeds whichcontain PaAos with low functionality. It is an object of that methodinvolves genotyping a first population of guayule plants or seeds thatcontain PaAos with low functionality, selecting from the firstpopulation one or more guayule plants or seeds containing PaAos with lowfunctionality based the genotyping, and producing from the selected oneor more guayule plants or seeds containing PaAos with low functionalitya second population of guayule plants or seeds containing PaAos with lowfunctionality. It is another object of this invention PaAos with lowfunctionality contains at least one amino acid selected from group ofN318, V408, W459, conservative amino acids substitutions thereof, andnon-conservative amino acid substitutions at S332, E336, R339, S359, andS411.

It is an object of this invention to have a method of identifying a highrubber producing guayule by detecting the presence of PaAos having aminoacids N318, V408 and/or W459, or conservative amino acid substitution atone or more of these positions and/or having non-conservative amino acidsubstitutions at S332, E336, R339, S359, and S411 in a test guayule. Itis further object of this invention that the method involves contactingthe PaAos from the test guayule with monoclonal antibodies that binds toPaAos having the above mentioned amino acids, and determining if themonoclonal antibodies bind to the PaAos from the test guayule, wherebinding indicates the presence of the high rubber producing PaAos and nobinding indicates the present of the low rubber producing PaAos. It isanother object of this invention that the invention involves obtainingnucleic acids from the test guayule, performing a PCR assay with theobtained nucleic acids, a label, and primer sets SEQ ID NOs: 22 and 23,and SEQ ID NOs: 24 and 25, or similar sequences that encode conservativeamino acid substitutions, and determining if an amplicon is generated;such that when an amplicon is produced, then the test guayule contains aPaAos nucleic acid sequence that encodes PaAos having amino acids N318,V408 and/or W459, or one or more of the conservative amino acidsubstitution at these positions, and/or non-conservative amino acidsubstitutions at S332, E336, R339, S359, and S411, and the test guayuleis a high rubber producing guayule.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the design of the plasmids used in Example 1 for theoverexpression of PaAos (pND6-Aos), silencing of PaAos (pND6-AosiL), andfor the control plasmid (pND6). Each expression vector features theNPTII gene to confer kanamycin resistance for selection, and the controlplasmid contains the GUS (β-glucuronidase) reporter gene instead ofPaAos or a portion of PaAos in reverse complementary orientation. Thus,one can use a histochemical GUS staining assay (the chromogenicsubstrate X-Gluc (C₁₄H₁₃BrCINO₇)) as a visual indicator of transformedtissues with the negative control plasmid.

FIG. 2 shows the primers used in the qRT-PCR, PCR reaction conditions,and the expected amplicon product size to determine RNA expression inthe genetically altered guayule. Amplicon (mRNA product) PaAos_(OE) isderived from plants transformed with pND6-Aos (Aos overexpression).Amplicon (mRNA product) PaAos_(RNAi) is derived from plants transformedwith pND6-AosiL (Aos silencing). Amplicon (mRNA product) 18S is from 18Sribosome RNA. Amplicon (mRNA product) PaAos is for wild-type guayuleplants with intact PaAos gene.

FIG. 3 provides size and weight measurements of the four types ofguayule plants grown under different conditions in growth chambers.Plants are initially transferred to soil from tissue culture media andgrown under greenhouse conditions for one month. Following, plants aremoved to controlled-temperature growth chamber conditions under 27° C.(16 h)/25° C. (8 h) and at 27° C. (16 h)/10° C. (8 h). The four type ofguayule plants are wild-type (G7-11.1 and G7-11.2), guayule transformedwith the empty expression vector pND6 (pND6-10, pND6-12, pND6-35),guayule transformed with pND6-AosiL for silencing PaAos via RNAi(pND6-AosiL₇₋₂, pND6-AosiL₈₋₁, pND6-AosiL₉₋₁₆, pND6-AosiL₁₂₋₁,), andguayule transformed with pND6-Aos to overexpress PaAos (pND6-Aos₄₋₁,pND6-Aos₄₋₂, pND6-Aos₅₋₁, pND6-Aos₇₋₁,) at 2 months old. G7-11 is abreeder's nomenclature for what later became the USDA publicly releasedguayule Germplasm line AZ-2 (Reg. No. GP-9; PI 599676). The biomass ofthe shoot (leaves plus stems) and root are weighed in 2 months oldguayule plants grown in growth chambers under 27° C. (16 h)/25° C. (8 h)and at 27° C. (16 h)/10° C. (8 h). The asterisks, (*), (**) and (***),indicate significant difference in comparison to (non-altered) G7-11 atp>0.05, 0.005 and 0.0005, respectively.

FIG. 4 shows SPAD values indicating leaf chlorophyll concentration(“SPAD units”) for wild-type guayule (G7-11), guayule transformed withthe empty expression vector (pND6); guayule transformed with pND6-Aos(Oe), and guayule transformed with pND6-AosiL (RNAi) grown at 27° C. (16h)/25° C. (8 h) or 27° C. (16 h)/10° C. (8 h).

FIG. 5 shows the number of branches and stem diameter of 2 months oldgenetically altered guayule (pND6-AosiL and pND6-Aos lines), non-alteredguayule (G7-11) and empty vector control guayule (pND6) plants grown ingrowth chambers at 27° C. (16 h)/25° C. (8 h) or at 27° C. (16 h)/10° C.(8 h). pND6-AosiL genotypes have larger number of stems than thenon-altered and empty vector controls. Additionally, the mature stembarktissues in pND6-AosiL have significantly thicker diameter (ranging from35% to 54%) under both 27° C. (16 h)/25° C. (8 h) and 27° C. (16 h)/10°C. (8 h).

FIG. 6 shows results from gel permeation chromatography for elution ofcyclohexane extractables from transformed and non-altered guayule plantlines. The natural rubber molecular weight is calculated using Astrasoftware for three pND6-AosiL transformed guayule plants, three pND6-Aostransformed guayule plants, two pND6 transformed guayule plants, andnon-altered guayule G7-11. The error bars represent 3 different plantswith 3 technical replicates.

FIG. 7 shows the relative expression of PaAos in guayule line G7-11,guayule line W6 549 (“W6549”), and guayule line PI 478652 (“478652”).

FIG. 8A, FIG. 8B, and FIG. 8C show single nucleotide polymorphisms(SNPs) in PaAos coding sequence for guayule cultivars W6 549 (“W6549”;SEQ ID NO: 12), G7-11 (SEQ ID NO: 9), and PI 478652 (“478652”; SEQ IDNO: 14). The SNPs are contained in boxes.

FIG. 9 shows an alignment of PaAos amino acid sequences obtained fromguayule cultivars W6 549 (“W6549”; SEQ ID NO: 13), PI 478652 (“478652”;SEQ ID NO: 15) and G7-11 (SEQ ID NO: 10). The boxes highlight thedifferent amino acids in the cultivars.

DETAILED DESCRIPTION OF THE INVENTION

This invention involves the discovery that a reduction in the amount offunctional PaAos in guayule results in an increase in the amount ofrubber produced. Further, different cultivars of guayule, havingdifferent DNA and amino acid sequences for PaAos and PaAos,respectively, produce different amounts of rubber. As such, one candistinguish between guayule cultivars that are “high” rubber producersand “low” rubber producers based on the differences in the DNA and/oramino acid sequence of PaAos and/or PaAos. Because single nucleotidepolymorphisms (SNPs) exist in these different cultivars, one can useprimers and PCR techniques to determine if any particular guayule is a“high” rubber producing guayule or a “low” rubber producing guayule.Alternatively, one can use antibodies that bind to the different PaAosproteins to determine if any particular guayule is a “high” rubberproducer or a “low” rubber producer. As discussed below, guayulecultivar W6 549 has the lowest average rubber content (%) and guayulecultivar PI 478652 has the highest average rubber content, among thetested cultivars. PaAos from both cultivars have slight differences inDNA sequences which, along with the differences in the amino acidsequences, can be used to determine if any particular cultivar is a highor low rubber producer. Any guayule that produces PaAos withconservative amino acid changes to SEQ ID NO: 15 is a high rubberproducing guayule; any guayule that produces PaAos with conservativeamino acid changes to SEQ ID NO: 13 is a low rubber producing guayule.Thus, one can screen plants for PaAos with conservative sequences to SEQID NO: 15 via DNA or protein assays. Alternatively, one screening forguayule with PaAos with the similar (or lower) level of functionality asguayule cultivar PI 478652's PaAos would identify a high rubberproducing guayule; whereas screening for guayule with PaAos with ahigher level of functionality would identify a low rubber producingguayule.

Because changes in PaAos functionality changes the amount of rubberproduced by guayule, this invention also involves increasing a guayule'srubber production by reducing the amount of functional PaAos present inthe genetically altered guayule (compared to the amount of functionalPaAos present in non-altered guayule). In one embodiment, thegenetically altered guayule has a mutation in PaAos, such as, a nullmutation which results in (i) no protein is produced, (ii) a truncatedprotein is produced which has no functionality, or (iii) a full-lengthprotein is produced which has no functionality. Other mutations simplyreduce the functionality (activity) of PaAos. Non-limiting examples ofmutations that reduce or eliminate PaAos functionality include (i)changing a codon encoding an amino acid to a stop codon (see Table 1supra for the sequence of stop codons), (ii) changing the translationinitiation codon (ATG) to any other codon to disrupt proteintranslation, (iii) changing a ribosome binding site's sequence todisrupt protein translation, (iv) changing one or more splice sitecodons to alter protein sequence, (v) deleting some or all of the gene'sDNA sequence, (vi) inserting DNA into the gene, and (vii) changing oneor more DNA codon sequences to encode non-conservative amino acids.Within SEQ ID NO: 9, nucleotides 34-36 encode ATG, the translationinitiation codon for G7-11 guayule. Similarly, nucleotides 1-3 of SEQ IDNOs: 12 and 14 encode ATG, the translation initiation codon forcultivars PI 478652 and W6549, respectfully. Thus, a change in thenucleotide sequence of the equivalent codon in any other guayule wouldhave the same result. Within PaAos, non-conservative amino acidsubstitutions at D318, S332, E336, R339, S359, I408, S411, and/or L459(to name a few) result in PaAos with reduced functionality. One altersguayule DNA using the methods described herein and assesses changes inPaAos functionality via the methods described herein (e.g., assessingthe amount of rubber produced by the altered guayule) or using methodsknown to one of ordinary skill in the art. One can utilize SNPs,antibodies, and other methods to identify the guayule that encode thealtered amino acids. When no functional PaAos is produced or when PaAoswith reduced functionality is produced, then it is understood that thealtered guayule produces “a reduced amount of functional PaAos”. Suchaltered guayule producing a reduced amount of functional PaAos isanother embodiment of this invention.

Because this invention involves production of genetically altered plantsand involves recombinant DNA techniques, the following definitions areprovided to assist in describing this invention. The terms “isolated”,“purified”, or “biologically pure” as used herein, refer to materialthat is substantially or essentially free from components that normallyaccompany the material in its native state or when the material isproduced. In an exemplary embodiment, purity and homogeneity aredetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A nucleicacid or particular bacteria that are the predominant species present ina preparation is substantially purified. In an exemplary embodiment, theterm “purified” denotes that a nucleic acid or protein that gives riseto essentially one band in an electrophoretic gel. Typically, isolatednucleic acids or proteins have a level of purity expressed as a range.The lower end of the range of purity for the component is about 60%,about 70% or about 80% and the upper end of the range of purity is about70%, about 80%, about 90% or more than about 90%.

The term “gene” refers to a DNA sequence involved in producing a RNA orpolypeptide or precursor thereof. The polypeptide or RNA is encoded by afull-length coding sequence (cds) or by intron-interrupted portions ofthe coding sequence, such as exon sequences. In one embodiment of thisinvention, the gene involved is Parthenium argentatum allene oxidesynthase (PaAos or Aos). PaAos cDNA and amino acid sequence is found inGenBank accession number X78166.2 which is cultivar G7-11(wild-type/non-altered) (USDA publicly released guayule Germplasm lineAZ-2 (Reg. No. GP-9; PI 599676)). The cDNA sequence is in SEQ ID NO: 9;the protein sequence is in SEQ ID NO: 10. SEQ ID NO: 12 is the cDNAsequence for PaAos and SEQ ID NO: 13 is the amino acid sequence forPaAos in guayule W6549 cultivar. SEQ ID NO: 14 is the cDNA sequence forPaAos and SEQ ID NO: 15 is the amino acid sequence for PaAos in guayule478652 cultivar. A molecular marker uses SNPs within PaAos todifferentiate the cultivars with differences in PaAos amino acidsequences which result in PaAos with different functionalities.

The term “nucleic acid” as used herein, refers to a polymer ofribonucleotides or deoxyribonucleotides. Typically, “nucleic acid”polymers occur in either single- or double-stranded form, but are alsoknown to form structures comprising three or more strands. The term“nucleic acid” includes naturally occurring nucleic acid polymers aswell as nucleic acids comprising known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Exemplary analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs). “DNA”, “RNA”, “polynucleotides”, “polynucleotidesequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleicacid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and“isolated nucleic acid fragment” are used interchangeably herein.

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp), or nucleotides (nt). Estimates are typically derived fromagarose or acrylamide gel electrophoresis, from sequenced nucleic acids,or from published DNA sequences. For proteins, sizes are given inkiloDaltons (kDa) or amino acid residue numbers. Proteins sizes areestimated from gel electrophoresis, from sequenced proteins, fromderived amino acid sequences, or from published protein sequences.

Unless otherwise indicated, a particular nucleic acid sequence for eachamino acid substitution (alteration) also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), the complementary (or complement) sequence, and thereverse complement sequence, as well as the sequence explicitlyindicated. Specifically, degenerate codon substitutions may be achievedby generating sequences in which the third position of one or moreselected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); andRossolini et al., Mol. Cell. Probes 8:91-98(1994)). Because of thedegeneracy of nucleic acid codons, one can use various differentpolynucleotides to encode identical polypeptides. Table 1, infra,contains information about which nucleic acid codons encode which aminoacids and is useful for determining the possible nucleotidesubstitutions that are included in this invention.

TABLE 1 Amino acid Nucleic acid codons Ala/A GCT, GCC, GCA, GCG Arg/RCGT, CGC, CGA, CGG, AGA, AGG Asn/N AAT, AAC Asp/D GAT, GAC Cys/CTGT, TGC Gln/Q CAA, CAG Glu/E GAA, GAG Gly/G GGT, GGC, GGA, GGG His/HCAT, CAC Ile/I ATT, ATC, ATA Leu/L TTA, TTG, CTT, CTC, CTA, CTG Lys/KAAA, AAG Met/M ATG Phe/F TTT, TTC Pro/P CCT, CCC, CCA, CCG Ser/STCT, TCC, TCA, TCG, AGT, AGC Thr/T ACT, ACC, ACA, ACG Trp/W TGG Tyr/YTAT, TAC Val/V GTT, GTC, GTA, GTG Stop TAA, TGA, TAG

In addition to the degenerate nature of the nucleotide codons whichencode amino acids, alterations in a polynucleotide that result in theproduction of a chemically equivalent amino acid at a given site, but donot affect the functional properties of the encoded polypeptide, arewell known in the art. “Conservative amino acid substitutions” are thosesubstitutions that are predicted to interfere least with the propertiesof the reference polypeptide. In other words, conservative amino acidsubstitutions substantially conserve the structure and the function ofthe reference protein. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine or histidine,is also expected to produce a functionally equivalent protein orpolypeptide. Table 2 provides a list of exemplary conservative aminoacid substitutions. Conservative amino acid substitutions generallymaintain (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a beta sheet or alpha helicalconformation, (b) the charge or hydrophobicity of the molecule at thesite of the substitution, and/or (c) the bulk of the side chain. Inanother embodiment, groups of amino acids that are conservativesubstitutions for each other are (i) alanine (Ala or A), serine (Ser orS), and threonine (Thr or T); (ii) aspartic acid (Asp or D) and glutamicacid (Glu or E); (iii) asparagine (Asn or N) and glutamine (Gln or Q);(iv) arginine (Arg or R) and lysine (Lys or K); (v) isoleucine (Ile orI), leucine (Leu or L), methionine (Met or M), and valine (Val or V);and (vi) phenylalanine (Phe or F), tyrosine (Tyr or Y), and tryptophan(Trp or W). See, Creighton, Proteins, W.H. Freeman and Co. (1984),contents of which are expressly incorporated herein. In yet anotherembodiment, amino acid(s) that are conservative substitutes for oneamino acid are grouped by the following characteristics: aliphatic aminoacids (alanine, glycine, isoleucine, leucine, and valine); hydroxyl orsulfur containing amino acids (cysteine, serine, methionine, andthreonine); cyclic (proline); aromatic (phenylalanine, tryptophan, andtyrosine); basic (arginine, histidine, and lysine); acidic (aspartateand glutamate); and uncharged (asparagine and glutamine). As discussedbelow, wild-type guayule can be “high” rubber producers or “low” rubberproducers. In both types of guayule, there are several amino acidchanges in PaAos that be used to distinguish the “high” and “low” rubberproducers. As such, one may change DNA encoding one, two, or more of theamino acids to change a “low” rubber producing guayule into a “high”rubber producing guayule. Alternatively, one may change the DNA toencode an amino acid that is a conservative substitute (per Table 2) ofthe one, two, or more amino acids different in the “high” rubberproducing guayule. Further, because the “high” rubber producing guayulediscussed below have several amino acids different in PaAos than the“low” rubber producing guayule, the invention also includes anyconservative amino acids changes that can be made in these one, two, ormore amino acids.

TABLE 2 Amino Acid Conservative Substitute Ala Gly, Ser Arg His, Lys AsnAsp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln,His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Ile, Leu Phe His, Leu, Met, Trp, Tyr Ser Cys, Thr Thr Ser,Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

The term “primer” refers to an oligonucleotide which may act as a pointof initiation of DNA extension. A primer may occur naturally, as in apurified restriction digest, or may be produced synthetically.

A primer is selected to be “substantially complementary” to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence is sufficiently complementarywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

Oligonucleotides and polynucleotides that are not commercially availablecan be chemically synthesized e.g., according to the solid phasephosphoramidite triester method first described by Beaucage andCaruthers, Tetrahedron Letts. 22:1859-1862 (1981), or using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.12:6159-6168 (1984). Other methods for synthesizing oligonucleotides andpolynucleotides are known in the art. Purification of oligonucleotidesis by either native acrylamide gel electrophoresis or by anion-exchangeHPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

The terms “identical” or percent “identity”, in the context of two ormore polynucleotides or polypeptide sequences, refer to two or moresequences or sub-sequences that are the same or have a specifiedpercentage of nucleotides or amino acids (respectively) that are thesame (e.g., 80%, 85% identity, 90% identity, 99%, or 100% identity),when compared and aligned for maximum correspondence over a designatedregion as measured using a sequence comparison algorithm or by manualalignment and visual inspection.

The phrase “high percent identical” or “high percent identity”, in thecontext of two polynucleotides or polypeptides, refers to two or moresequences or sub-sequences that have at least about 80%, identity, atleast about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide or amino acididentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.In an exemplary embodiment, a high percent identity exists over a regionof the sequences that is at least about 16 nucleotides or amino acids inlength. In another exemplary embodiment, a high percent identity existsover a region of the sequences that is at least about 50 nucleotides oramino acids in length. In still another exemplary embodiment, a highpercent identity exists over a region of the sequences that is at leastabout 100 nucleotides or amino acids or more in length. In one exemplaryembodiment, the sequences are high percent identical over the entirelength of the polynucleotide or polypeptide sequences.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters may be used, or alternative parameters designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison is conducted, e.g., by the local homology algorithm of Smith& Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignmentalgorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by thesearch for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci.USA 85:2444 (1988), by computerized implementations of variousalgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), and/or by manual alignment and visual inspection (see, e.g.,Ausubel et al. (eds.), Current Protocols in Molecular Biology, 1995supplement).

The “complement” of a particular polynucleotide sequence is thatnucleotide sequence which would be capable of forming a double-strandedDNA or RNA molecule with the represented nucleotide sequence, and whichis derived from the represented nucleotide sequence by replacing thenucleotides by their complementary nucleotide according to Chargaff'srules (A< >T; G< >C) and reading in the 5′ to 3′ direction, i.e., inopposite direction of the represented nucleotide sequence (reversecomplement).

In one embodiment of the invention, sense and antisense RNAs and dsRNAcan be separately expressed in-vitro or in-vivo. In-vivo production ofsense and antisense RNAs may use different chimeric polynucleotideconstructs using the same or different promoters or using an expressionvector containing two convergent promoters in opposite orientation. Thesense and antisense RNAs which are formed (e.g., in the same host cellsor synthesized) then combine to form dsRNA. To be clear, wheneverreference is made herein to a dsRNA chimeric or fusion polynucleotide ora dsRNA molecule, that such dsRNA formed (e.g., in plant cells) fromsense and antisense RNA produced separately is also included. Also,synthetically made dsRNA and self-annealing RNA strands are includedherein when the sense and antisense strands are present together.

As used herein, the term “promoter” refers to a polynucleotide that, inits native state, is located upstream or 5′ to a translational startcodon of an open reading frame (or protein-coding region) and that isinvolved in recognition and binding of RNA polymerase and other proteins(trans-acting transcription factors) to initiate transcription. A “plantpromoter” is a native or non-native promoter that is functional in plantcells, even if the promoter is present in a microorganism that infectsplants or a microorganism that does not infect plants. The promotersthat are predominately functional in a specific tissue or set of tissuesare considered “tissue-specific promoters”. A plant promoter can be usedas a 5′ regulatory element for modulating expression of a particularlydesired polynucleotide (heterologous polynucleotide) operably linkedthereto. When operably linked to a transcribable polynucleotide, apromoter typically causes the transcribable polynucleotide to betranscribed in a manner that is similar to that of which the promoter isnormally associated.

Plant promoters include promoters produced through the manipulation ofknown promoters to produce artificial, chimeric, or hybrid promoters.Such promoters can also combine cis-elements from one or more promoters,for example, by adding a heterologous regulatory element to an activepromoter with its own partial or complete regulatory elements. The term“cis-element” refers to a cis-acting transcriptional regulatory elementthat confers an aspect of the overall control of gene expression. Acis-element may function to bind transcription factors, trans-actingprotein factors that regulate transcription. Some cis-elements bind morethan one transcription factor, and transcription factors may interactwith different affinities with more than one cis-element.

The term “vector” refers to DNA, RNA, a protein, or polypeptide that areto be introduced into a host cell or organism. The polynucleotides,protein, and polypeptide which are to be introduced into a host may betherapeutic or prophylactic in nature; may encode or be an antigen; maybe regulatory in nature; etc. There are various types of vectorsincluding viruses, viroids, plasmids, bacteriophages, cosmids, andbacteria.

An expression vector is nucleic acid capable of replicating in aselected host cell or organism. An expression vector can replicate as anautonomous structure, or alternatively integrate, in whole or in part,into the host cell chromosomes or the nucleic acids of an organelle, orit is used as a shuttle for delivering foreign DNA to cells, and thusreplicate along with the host cell genome. Thus, an expression vectorare polynucleotides capable of replicating in a selected host cell,organelle, or organism, e.g., a plasmid, virus, artificial chromosome,nucleic acid fragment, and for which certain genes on the expressionvector (including genes of interest) are transcribed and translated intoa polypeptide or protein within the cell, organelle or organism; or anysuitable construct known in the art, which comprises an “expressioncassette”. In contrast, as described in the examples herein, a“cassette” is a polynucleotide containing a section of an expressionvector. The use of the cassettes assists in the assembly of theexpression vectors. An expression vector is a replicon, such as plasmid,phage, virus, chimeric virus, or cosmid, and which contains the desiredpolynucleotide sequence operably linked to the expression controlsequence(s).

A heterologous polynucleotide sequence is operably linked to one or moretranscription regulatory elements (e.g., promoter, terminator and,optionally, enhancer) such that the transcription regulatory elementscontrol and regulate the transcription and/or translation of thatheterologous polynucleotide sequence. A cassette has the heterologouspolynucleotide operably linked to one or more transcription regulatoryelements. As used herein, the term “operably linked” refers to a firstpolynucleotide, such as a promoter, connected with a secondtranscribable polynucleotide, such as a gene of interest, where thepolynucleotides are arranged such that the first polynucleotide affectsthe transcription of the second polynucleotide. In some embodiments, thetwo polynucleotide molecules are part of a single contiguouspolynucleotide. In other embodiments, the two polynucleotides areadjacent. For example, a promoter is operably linked to a gene ofinterest if the promoter regulates or mediates transcription of the geneof interest in a cell. Similarly, a terminator is operably linked to thepolynucleotide of interest if the terminator regulates or mediatestranscription of the polynucleotide of interest, and in particular, thetermination of transcription. Constructs of the present invention wouldtypically contain a promoter operably linked to a transcribablepolynucleotide operably linked to a terminator.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, organism,nucleic acid, protein or vector, has been altered by the introduction ofa heterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell soaltered. Thus, for example, recombinant cells may expressgenes/polynucleotides that are not found within the native(non-recombinant or non-altered or wild-type) form of the cell orexpress native genes in an otherwise abnormal amount—over-expressed,under-expressed or not expressed at all—compared to the non-altered cellor organism. In particular, one alters the genomic DNA of a non-alteredplant by molecular biology techniques that are well-known to one ofordinary skill in the art and generate a recombinant plant.

The terms “transgenic”, “transformed”, “transformation”, and“transfection” are similar in meaning to “recombinant” “Transformation”,“transgenic”, and “transfection” refer to the transfer of apolynucleotide into a host organism or into a cell. Such a transfer ofpolynucleotides may result in genetically stable inheritance of thepolynucleotides or in the polynucleotides remaining extra-chromosomally(not integrated into the chromosome of the cell). Genetically stableinheritance may potentially require the transgenic organism or cell tobe subjected for a period of time to one or more conditions whichrequire the transcription of some or all of the transferredpolynucleotide in order for the transgenic organism or cell to liveand/or grow. Polynucleotides that are transformed into a cell but arenot integrated into the host's chromosome remain as an expression vectorwithin the cell. One may need to grow the cell under certain growth orenvironmental conditions in order for the expression vector to remain inthe cell or the cell's progeny. Further, for expression to occur theorganism or cell may need to be kept under certain conditions.Genetically altered organisms or cells containing the recombinantpolynucleotide are referred to as “transgenic” or “transformed”organisms or cells or simply as “transformants”, as well as recombinantorganisms or cells.

A genetically altered organism is any organism with any changes to itsgenetic material involving the invention described herein, whether inthe nucleus or cytoplasm (organelle). As such, a genetically alteredorganism may be a recombinant or transformed organism. A geneticallyaltered organism may also be an organism that was subjected to one ormore mutagens or the progeny of an organism that was subjected to one ormore mutagens and has mutations in its DNA caused by the one or moremutagens, as compared to the wild-type organism (i.e., organism notsubjected to the mutagens) or the non-altered organism (i.e., one thatcontains alterations that are not the subject matter of this invention).Also, an organism that has been bred to incorporate a mutation into itsgenetic material is a genetically altered organism.

The term “altered” means that a change occurred compared to the“non-altered” item. However, a “non-altered” item could contain changesthat are induced by man, but those changes are not the subject matter ofthe inventions described herein. For example, a non-altered guayulecontains none of the described genetic changes nor has been treated withany of the described external substance, but may contain pre-existingchanges which are not part of this invention. An altered guayule (whichalso is a genetically altered guayule) may contain DNA mutations whichchange PaAos' amino acid sequence, even if that sequence exists in anon-altered plant. Such DNA mutations may be induced by a mutagen (EMS,UV light, other radiation, etc.).

Transformation and generation of genetically altered monocotyledonousand dicotyledonous plant cells is well known in the art. See, e.g.,Weising, et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Pat. No.5,679,558; Agrobacterium Protocols, ed: Gartland, Humana Press Inc.(1995); and Wang, et al. Acta Hort. 461:401-408 (1998). A method togenerate genetically altered guayule is described in U.S. Pat. No.9,018,449 (Dong & Cornish) and in Dong, et al., Plant Cell Reports25:26-34 (2006). A method to generate transplastomic guayule is providedin U.S. Patent Application Publication 2014/0325699, contents of whichare expressly incorporated herein. The choice of method varies with thetype of plant to be transformed, the particular application and/or thedesired result. The appropriate transformation technique is readilychosen by the skilled practitioner.

A polynucleotide encoding PaAos (SEQ ID NOs: 9, 12, and/or 14), thereverse complement of PaAos, or a portion thereof (e.g., SEQ ID NO: 11),operably linked to one or two appropriate promoters, can be stablyinserted in a conventional manner into the genome (cytoplasmic genome ornucleic genome) of a single plant cell, and the altered plant cell canbe used in a conventional manner to produce a genetically altered plantthat produces the dsRNA of this invention. In this regard, a disarmedTi-plasmid, containing the polynucleotide of this invention, inAgrobacterium tumefaciens can be used to genetically alter the plantcell, and thereafter, a genetically altered plant can be regeneratedfrom the genetically altered plant cell using the procedures describedin the art, for example, in EP 0 116 718, EP 0 270 822, WO 84/02913 andEP 0 242 246. Plant regeneration from cultured protoplasts is describedin Evans et al., Protoplasts Isolation and Culture, in Handbook of PlantCell Culture, pp. 124-176, MacMillan Publishing Company, New York, 1983;and Binding, Regeneration of Plants, in Plant Protoplasts, pp. 21-73,CRC Press, Boca Raton, 1985. Regeneration can also be obtained fromplant callus, explants, organs, or parts thereof. Such regenerationtechniques are described generally in Klee, et al., Ann. Rev. of PlantPhys. 38:467-486 (1987).

Preferred Ti-plasmid vectors each contain the polynucleotides describedherein between the border sequences, or at least located to the left ofthe right border sequence, of the T-DNA of the Ti-plasmid. Of course,other types of vectors may be used to transform the plant cell, usingprocedures such as direct gene transfer (as described, for example in EP0 233 247), pollen mediated transformation (as described, for example inEP 0 270 356, WO 85/01856, and U.S. Pat. No. 4,684,611), plant RNAvirus-mediated transformation (as described, for example in EP 0 067 553and U.S. Pat. No. 4,407,956), liposome-mediated transformation (asdescribed, for example in U.S. Pat. No. 4,536,475), and other methodssuch as the methods for transforming certain lines of corn (e.g., U.S.Pat. No. 6,140,553; Fromm, et al., Bio/Technology 8:833-839 (1990);Gordon-Kamm, et al., The Plant Cell 2:603-618 (1990) and rice(Shimamoto, et al., Nature 338:274-276 (1989); Datta et al.,Bio/Technology 8:736-740 (1990)) and the method for transformingmonocots generally (WO 92/09696). For cotton transformation, the methoddescribed in WO 2000/71733 can be used. For soybean transformation,reference is made to methods known in the art, e.g., Hinchee, et al.(Bio/Technology 6:915 (1988)) and Christou, et al. (Trends Biotechnology8:145 (1990)) or the method of WO 00/42207.

The resulting genetically altered plant can be used in a conventionalplant breeding scheme to produce more genetically altered plants withthe same characteristics or to introduce the polynucleotide into othervarieties of the same or related plant species. Seeds, which areobtained from the genetically altered plants, contain the expressionvector as a stable genomic insert. Altered plants include plants havingor derived from root stocks of plants containing the expression vector.Hence, any non-altered grafted plant parts inserted on a geneticallyaltered plant or plant part are included in the invention.

For a genetically altered plant that produces dsRNA, one constructs anexpression vector or cassette (made from DNA) that encodes, at aminimum, a first promoter and the dsRNA sequence of interest such thatthe promoter sequence is 5′ (upstream) to and operably linked to thedsRNA sequence. The expression vector or cassette may optionally containa second promoter (same as or different from the first promoter)upstream and operably linked to the reverse complementary sequence ofthe dsRNA sequence such that two strands of RNA that are complementaryto each other are produced. Alternatively, the expression vector orcassette can contain one promoter operably linked to both the dsRNAsequence (sense strand) in question and the complement or reversecomplement of the dsRNA sequence (anti-sense strand) in question, suchthat the transcribed RNA bends on itself and the two desires sequencesanneal. Alternatively, a second expression vector or cassette (made fromDNA) may encode, at a minimum, a second promoter (same as or differentfrom the promoter) operably linked to the reverse complementary sequenceof the dsRNA such that two strands of complementary RNA are produced inthe plant. The expression vector(s) or cassette(s) is/are inserted in aplant cell genome (nuclear or cytoplasmic). The promoter(s) used shouldbe a promoter(s) that is/are active in a plant and is/are heterologousto PaAos (not normally driving the transcription of RNA of genomicPaAos). Of course, the expression vector or cassette may have othertranscription regulatory elements, such as enhancers, terminators, etc.

Promoters (and more specifically, heterologous promoters for PaAos) thatare active in plants are well-known in the field. Such promoters may beconstitutive, inducible, and/or tissue-specific. Non-limiting examplesof constitutive plant promoters include 35S promoters of the cauliflowermosaic virus (CaMV) (e.g., of isolates CM 1841 (Gardner, et al., NucleicAcids Research 9:2871-2887 (1981)), CabbB-S (Franck, et al., Cell21:285-294 (1980)) and CabbB-JI (Hull and Howell, Virology 86:482-493(1987))), ubiquitin promoter (e.g., the maize ubiquitin promoter ofChristensen, et al., Plant Mol. Biol. 18:675-689 (1992)), gos2 promoter(de Pater, et al., The Plant J. 2:834-844 (1992)), emu promoter (Last,et al., Theor. Appl. Genet. 81:581-588 (1990)), actin promoter (see,e.g., An, et al., The Plant J. 10:107 (1996)) and Zhang, et al., ThePlant Cell 3:1155-1165 (1991)); Cassava vein mosaic virus promoters(see, e.g., WO 97/48819 and Verdaguer, et al., Plant Mol. Biol.37:1055-1067 (1998)), the pPLEX series of promoters from SubterraneanClover Stunt Virus (WO 96/06932, particularly the S4 or S7 promoter),alcohol dehydrogenase promoter (e.g., pAdh1S (GenBank accession numbersX04049, X00581)), and the TR1′ promoter and the TR2′ promoter whichdrive the expression of the 1′ and 2′ genes, respectively, of the T-DNA(Velten, et al., EMBO J. 3:2723-2730 (1984)). Tissue-specific promotersare promoters that direct a greater level of transcriptional expressionin some cells or tissues of the plant than in other cells or tissue.Non-limiting examples of tissue-specific promoters include thephosphoenolpyruvate carboxylase (PEP or PPC1) promoter (Pathirana, etal., Plant J. 12:293-304 (1997), and Kausch, et al., Plant Mol. Biol.45(1):1-15 (2001)), chlorophyll A/B binding protein (CAB) promoter(Bansal, et al., Proc. Natl. Acad. Sci. USA 89(8):3654-8 (1992)), smallsubunit of ribulose-1,5-bisphosphate carboxylase (ssRBCS) promoter(Bansal, et al., Proc. Natl. Acad. Sci. USA 89(8):3654-8 (1992)),senescence activated promoter (SEE1) (Robson, et al., Plant Biotechnol.J. 2(2):101-12 (2004)), and sorghum leaf primoridia specific promoter(RS2) (GenBank Accession No. E1979305.1). These promoters (PPC1, CAB,ssRBCS, SSE1, and RS2) are all active in the aerial part of a plant.Further, the PPC1 promoter is a strong promoter for expression invascular tissue. Some examples of phloem specific promoters are thesucrose synthase-1 promoters (CsSUS1p and CsSUS1p-2) (Singer et al.,Planta 234:623-637 (2011)) and the phloem protein-2 promoter (CsPP2)(Miyata et al., Plant Cell Report 31(11):2005-2013 (2012)) from Citrussinensis. Alternatively, a plant-expressible promoter may also be awound-inducible promoter, such as the promoter of the pea cell wallinvertase gene (Zhang, et al., Plant Physiol. 112:1111-1117 (1996)).

Other types of RNA polymerase promoters that may be used are promotersfrom microorganisms, such as, but not limited to the bacteriophage T7RNA polymerase promoter, yeast Galactose (GAL1) promoter, yeastglyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, yeast AlcoholOxidase (AOX) promoter.

Other elements used to increase transcription expression in plant cellsinclude, but are not limited to, an intron (e.g., hsp70 intron) at the5′ end or 3′ end of the chimeric gene, or in the coding sequence of thechimeric dsRNA gene (such as, between the region encoding the sense andantisense portion of the dsRNA), promoter enhancer elements, duplicatedor triplicated promoter regions, 5′ leader sequences different from thechimeric gene or different from an endogenous (plant host) gene leadersequence, 3′ untranslated sequences different from the chimeric gene ordifferent from an endogenous (plant host) 3′ untranslated sequence.

The expression vector or cassette could contain suitable 3′ untranslatedtranscription regulation sequences (i.e., transcript formation andpolyadenylation sequences). Potential polyadenylation and transcriptformation sequences include those sequences in the nopaline synthasegene (Depicker, et al., J. Molec. Appl. Genetics 1:561-573 (1982)), theoctopine synthase gene (Gielen, et al., EMBO J. 3:835-845 (1984)), theSCSV or the Malic enzyme terminators (Schunmann, et al., PlantFunctional Biology 30:453-460 (2003)), and the T-DNA gene 7 (Velten andSchell, Nucleic Acids Research 13:6981-6998 (1985)).

The term “plant” includes whole plants, plant organs, progeny of wholeplants or plant organs, embryos, somatic embryos, embryo-likestructures, protocorms, protocorm-like bodies (PLBs), and suspensions ofplant cells. Plant organs comprise, e.g., shoot vegetativeorgans/structures (e.g., leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g., bracts, sepals, petals, stamens,carpels, anthers and ovules), seed (including embryo, endosperm, andseed coat) and fruit (the mature ovary), plant tissue (e.g., vasculartissue, ground tissue, and the like) and cells (e.g., guard cells, eggcells, trichomes and the like). The class of plants that can be used inthe method of the invention is generally as broad as the class of higherand lower plants amenable to the molecular biology and plant breedingtechniques described herein, specifically angiosperms (monocotyledonous(monocots) and dicotyledonous (dicots) plants). It includes plants of avariety of ploidy levels, including aneuploid, polyploid, diploid,haploid and hemizygous. The genetically altered plants described hereinare guayule plants.

Rubber yield may be expressed as a product of rubber content (% rubber)and biomass (dry weight/unit area). Thus, rubber yield may be improvedby increasing either biomass and/or rubber content. The altered guayuledescribed herein produce more rubber and have higher rubber content thannon-altered guayule, thereby increasing the processing efficiency of theguayule shrub.

Various methods exist to create a mutation. These methods are well-knownto one of ordinary skill in the art. One method is by transforming theplant with a plasmid containing 5′ sequence and 3′ sequence of the geneand allowing a cross-over event to occur, thereby excising the DNA fromthe plant's genome that is between the plasmid's 5′ sequence and 3′sequence. Also, one can use transposon-mediated mutation to delete oradd DNA to PaAos which would result in the encoded protein having areduced functionality compared to a non-altered PaAos. Two other methodsinvolve using a chemical mutagen (such as ethyl methanesulfonate (EMS))or physical agents (radiation, UV, or proton, for example) to generategenetic mutations in plant cells and/or germplasm. Also, one may useTALEN or CRISPR-Cas9 to mutate the sequence of the target gene (PaAos)such that a desired mutation is generated. One of ordinary skill in theart can also use targeted cleavage events to induce targetedmutagenesis, induce targeted deletions of cellular DNA sequences, andfacilitate targeted recombination and integration at predeterminedchromosomal locations to generate one or more of the null mutationsdiscussed above or to reduce the mutated protein's functionality.Nucleotide editing techniques are well-known and described in Urnov, etal., Nature 435(7042):646-51 (2010); U.S. Patent Publications2003/0232410, 2005/0208489, 2005/0026157, 2005/0064474, 2006/0188987,2009/0263900, 2009/0117617, 2010/0047805, 2011/0207221, 2011/0301073,2011/089775, 2011/0239315, and 2011/0145940; and InternationalPublication WO 2007/014275, the disclosures of which are incorporated byreference in their entireties for all purposes. Cleavage occurs by usingspecific nucleases such as engineered zinc finger nucleases (ZFN),transcription-activator like effector nucleases (TALENs), or using theCRISPR/Cas9 system with an engineered crRNA/tracr RNA (‘single guideRNA’) to guide specific cleavage. U.S. Patent Publication 2008/0182332describes the use of non-canonical zinc finger nucleases (ZFNs) fortargeted modification of plant genomes; U.S. Patent Publication2009/0205083 describes ZFN-mediated targeted modification of a plantEPSPS locus; U.S. Patent Publication 2010/0199389 describes targetedmodification of a plant Zp15 locus and U.S. Patent Publication No.20110167521 describes targeted modification of plant genes involved infatty acid biosynthesis. In addition, Moehle, et al., Proc. Natl. Acad.Sci. USA 104(9):3055-3060 (2007) describes using designed ZFNs fortargeted gene addition at a specified locus. U.S. Patent Publication2011/0041195 describes methods of making homozygous diploid organisms.Information on CRISPR/Cas9 system is found, e.g., aten.wikipedia.org/wiki/CRISPR;neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology;and Cong, et al., Science, 339:819-823 (2013). Sigma-Aldrich (St. Louis,Mo.) and Origene Technologies, Inc. (Rockville, Md.) are among thecompanies that sell CRISPR/Cas9 kits.

After using any of these various methods to induce genetic alterationsin a cell's genome, one induces the treated cells to grow into plantsand then screen the plants using the methods described herein for PaAoshaving reduced or no functionality, and/or for reduced amounts of PaAosor no PaAos (via reduction in gene expression and/or mRNA translationand/or other mechanism), and/or for elevated production of rubber(compared to amounts present in non-altered plants). Thus, anotherembodiment of this invention is the generation of altered guayule havinga genetic alteration in PaAos such that the altered guayule producesmore rubber than produced by non-altered guayule.

In another embodiment, one synergistically increases the amount ofrubber produced by exposing an altered guayule to cold temperatures(between approximately 7° C. and approximately 15° C. or betweenapproximately 10° C. and approximately 15° C.; approximately 8 hours perday) for approximately two weeks or more. The altered guayule maycontain one of more of the following alterations: (1) DNA encoding (i)anti-sense RNA for PaAos, (ii) double-stranded RNA for PaAos, (iii) amutation within PaAos that encodes a PaAos with reduced or no function;and/or (2) exogenously administered PaAos dsRNA. The combination of anyof the above alterations and exposure to cold temperatures (betweenapproximately 7° C. and approximately 15° C. or between approximately10° C. and approximately 15° C.; approximately 8 hours per day) forapproximately two weeks or more result in production of increasedamounts of rubber than produced by the non-altered plant exposed to thesame temperatures for the same time period.

Many techniques involving molecular biology discussed herein arewell-known to one of ordinary skill in the art and are described in,e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual 4th ed.2012, Cold Spring Harbor Laboratory; Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, 1994—current, John Wiley & Sons; andKriegler, Gene Transfer and Expression: A Laboratory Manual (1993).Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biologymaybe found in e.g., Benjamin Lewin, Genes IX, Oxford University Press,2007 (ISBN 0763740632); Krebs, et al. (eds.), The Encyclopedia ofMolecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN1-56081-569-8).

The terms “approximately” and “about” refer to a quantity, level, valueor amount that varies by as much as 30% in one embodiment, or in anotherembodiment by as much as 20%, and in a third embodiment by as much as10% to a reference quantity, level, value or amount. As used herein, thesingular form “a”, “an”, and “the” include plural references unless thecontext clearly dictates otherwise. For example, the term “a bacterium”includes both a single bacterium and a plurality of bacteria.

The term “nucleic acid consisting essentially of”, “polynucleotideconsisting essentially of”, and “RNA consisting essentially of”, andgrammatical variations thereof, means a polynucleotide that differs froma reference nucleic acid sequence by 20 or fewer nucleotides and alsoperform the function of the reference polynucleotide sequence. Suchvariants include sequences which are shorter or longer than thereference nucleic acid sequence, have different residues at particularpositions, or a combination thereof.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples and theaccompanying drawings, which are included herein only to furtherillustrate the invention and are not intended to limit the scope of theinvention as defined by the claims. The examples and drawings describeat least one, but not all embodiments, of the inventions claimed.Indeed, these inventions may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements.

Example 1. Construction of Genetically Altered Guayule

To better understand the role of PaAos in rubber synthesis, geneticallyaltered P. argentatum plants are generated in which either PaAos isover-expressed or PaAos is silenced by RNAi. The various plasmids usedto achieve the overexpression or silencing of PaAos in guayule are shownin FIG. 1. To generate these plasmids, the guayule Aos (PaAos) isamplified by PCR using genomic DNA as a template. The primers used toamplify PaAos are designed from the cDNA PaAos sequence published inNCBI database (GeneBank accession no. X78166.2) and have the followingsequences: forward primer 5′-cttaagaggtggtATGGACCCATCGTCTAAACCC-3′ (SEQID NO: 1) and reverse primer 5′-ggatccTCATATACTAGCTCTCTTCAGGG-3′ (SEQ IDNO: 2). The nucleotides in lower case and underlined in the forwardprimer are the recognition nucleotides for restriction enzyme AflII; thenucleotides in lower case and underlined in the reverse primer are therecognition nucleotides for restriction enzyme BamHI. The PCR cycleprogram is the following: 94° C. for 2 minutes (initial heating step)and PaAos is amplified at 40 cycles of 94° C. for 30 seconds(denaturation), 71° C. for 30 seconds (annealing) and 68° C. for 1minute (extension) and an additional 5 minutes extension at 68° C. Theresulting amplicon is purified and subcloned into pGEM T Easy vector(Promega, Madison, Wis.) using manufacturer's recommended protocol andsequenced to confirm the sequence of the plasmids. The cDNA sequence isin SEQ ID NO: 9 and the amino acid sequence is in SEQ ID NO: 10.Subsequently, the PaAos amplicon is cut using AflII and BamHI. PlasmidpND6 has a Nos promoter driving the NPTII gene for conferring kanamycinresistance and a potato ubiquitin promoter (Garbarino and Belknap, PlantMol. Biol. 24:119-127 (1994)) controlling the GUSplus gene (CambiaLabs,Canberra, Australia). See FIG. 1. Plasmid pND6-Aos (FIG. 1) is generatedby replacing the GUSplus gene in pND6 with cDNA PaAos sequence (SEQ IDNO: 9). Plasmid pND6-AosiL (FIG. 1) is generated by replacing theGUSplus gene in pND6 with an inverted repeat of a partial cDNA PaAossequence (SEQ ID NO: 11) containing a loop sequence of the Bar genebetween the inverted repeat of the partial cDNA PaAos sequence; thecomplete sequence replacing GUSplus is SEQ ID NO: 26. The plasmids pND6,pND6-AosiL, and pND6-Aos are used to transform Agrobacterium EHA101competent cells using the protocol described in Hood, et al., J.Bacteriol. 168:1291-1301 (1986).

The transformed Agrobacterium EHA101 either harboring pND6, pND6-AosiL,or pND6-Aos are used to transform guayule G7-11 using the protocols setforth below. See also Dong, et al. (2006) and Dong, et al., IndustrialCrops and Products 46:15-24 (2013). For Agrobacterium transformation,the overnight Agrobacterium culture are prepared by inoculating 50 μLglycerol stock into a 50 mL Falcon tube containing 5 mL LB medium plus40 mg/L rifampcin and 200 mg/L spectinomycin, and shaking at 200 rpm at28° C. The suspension then is centrifuged for 15 minutes at 1600×g atroom temperature. The supernatant is discarded, and the pellet isre-suspended in 25 mL of inoculation solution ( 1/10 MS salts plus BA (2mg/L), NAA (0.5 mg/L), glucose (10 g), acetosyringone (200 μM), pluronicF68 (0.05%), pH=5.2 (PhytoTechnology Labs, Shawnee Mission, Kans.)). Forleaf transformation, leaf sections are cut from the plants in theMagenta boxes (Caisson Labs, Smithfield, Utah). The adaxial side of eachleaf is placed facing up in a Petri dish containing 5 ml Agrobacteriumsuspension. The leaf is cut into ˜10 mm strips and immediately placed inan empty Petri dish in non-overlapping manner. When this Petri dish isfull, all leaf strips are blotted with the filter paper and placed intoanother empty Petri dish. The Petri dish is sealed by parafilm and leftin the dark at room temperature. The co-cultivation is replaced by thisco-desiccation according to Cheng, et al., In Vitro Cell Dev. Biol.Plant, 39, 595-604 (2003). After three days, leaf strips are transferredto MSB1T (MS medium with BA (1 mg/L), sucrose (30 g/L), phytagel (3g/L), and timentin (400 mg/L)) (Cheng, et al., Plant Cell Rep.,17(8):646-649 (1998)) for recovery at low light for 5 days. The leafstrips are then transferred to MSB1TK30 (MS medium containing BA (1mg/L), sucrose (30 g/L), phytagel (3 g/L), timentin (250 mg/L), andkanamycin (30 mg/L)) for selection under low light for two weeks. Theleaf strips are then subcultured every 2 weeks under high light tillgreen shoots emerged. Green shoots 10 mm and longer are transferred to½MS10.1TK10 for rooting (same as ½MSI0 but with timentin (250 mg/L) andkanamycin (10 mg/L)). After 2-4 weeks, the rooted plantlets aremicropropogated and subsequently transplanted into soil.

While the genetically altered guayule are still growing in tissueculture under selection, the genetically altered plants are screened forintegration of the expression vectors, pND6-Aos (PaAos in forwardorientation; SEQ ID NO: 9), pND6-AosiL (PaAos in the reverse orientation(a portion of reverse complement of PaAos is SEQ ID NO: 11)), and pND6(negative control). DNA is extracted from genetically altered plantsusing Sigma Kit (Sigma-Aldrich, St. Louis, Mo.). Approximately 150 mgleaf tissue (3 leaf tissues) are cut from the plants grown intissue-cultured, placed into 2 mL tubes and snapped-frozen in liquidnitrogen. A bead is added to pulverize the tissue into a fine powder ata frequency of 30/s for 1 minute using the mixer mill MM 400 tissuelyser (Verder Scientific, Inc., Newtown, Pa.).

PCR is carried out in 50 μL mixture containing Taq 2× Master Mix (NewEngland Biolabs, Ipswich, Mass.), 200 ng guayule genomic DNA or 20 pgplasmid DNA, and 100 ng of PaAos specific primers; namely SEQ ID NOs: 1and 2 for guayule transformed with pND6-Aos; and SEQ ID NOs: 3 and 4 forguayule transformed with pND6-AosiL. See FIG. 2. After heating thesamples to 94° C. for 2 minutes, the reaction proceeds with 35 cycles of94° C. for 30 seconds, 71° C. to amplify the PaAos in the overexpressionlines (pND6-Aos) for 30 seconds or 56° C. for the PaAos in the RNAilines (pND6-AosiL) for 30 seconds, and 68° C. for 1 minute. A finalelongation step is carried out at 68° C. for 5 minutes. PCR products areseparated by electrophoresis on a 1% (w/v) agarose gel. The band for theoverexpression lines is at ˜1.4 kbp, as expected; the band for the RNAilines is at ˜0.5 kbp as expected. The genetically altered guayule plantsharboring the empty plasmid (pND6 (negative control)) are confirmed byGUS staining (Karcher, S., ABLE 23:29-42 (2002)). Briefly, plant tissuesare placed in a 50 mL tube containing GUS assay solution (1 mM X-Gluc(5-bromo-4-chloro-3-indolyl) B-D-glucuronic acid in 50 mM Na₂HPO₄, pH7.0 and 0.1% Triton X-100). The reaction is incubated at 37° C. for 1hour followed by washing for 30 minutes with 70% ethanol to extract thechlorophyll.

Example 2. Determination of RNA Expression Levels in Genetically AlteredPlants

Guayule containing intact PaAos (non-altered; G7-11) and geneticallyaltered guayule containing one of the plasmids (pND6, pND6-Aos, orpND6-AosiL) are further screened to determine the RNA level (see Table3). Leaves from the various genetically modified plants (which are grownin tissue culture) are collected and snap-frozen in liquid nitrogen forRNA extraction. RNA is extracted using TRIzol® according tomanufacturer's recommended protocol (Ambion, Pittsburgh, Pa.). RNAconcentration is quantified with the NanoDrop ND1000 (ThermoScientific,Wilminton, Del.). RNA cleanup is performed using the RNeasy MinEluteCleanup kit according to manufacturer's recommended protocol (QiagenInc., Valencia, Calif.). The RNA is eluted with 30-50 μL of RNase-freewater along with on-column DNase1 treatment.

Using the RNA isolated from the leaves of the genetically alteredplants, cDNA is generated using iScript cDNA synthesis kit (Bio-Rad,Hercules, Calif.) according to the manufacturer's recommended protocolfor semi-quantitative PCR and real-time quantitative PCR (qRT-PCR). Anamount of 1 μg of RNA is used in the 20 mL reaction mixture. ForqRT-PCR, 2 μL of the diluted cDNA (1:20) is used in a 15 μL reactionmixture. In the qRT-PCR volume, 7.5 mL of iQ SYBR® Green Supermix isused (Bio-Rad, Hercules, Calif.). The qRT-PCR is run using the 7500 FastReal-Time PCR system (Applied Biosystem, Waltham, Mass.) with thefollowing thermal cycle: 95° C. pre-incubation for 3 minutes;amplification is performed for 40 cycles at 95° C. for 15 seconds and at60° C. for 30 seconds; the dissociation stage is set for 95° C. for 15seconds, at 60° C. for 1 minute, and at 95° C. for 15 seconds. EachqRT-PCR run is performed with three independent tissue samples, eachsample having two technical replicates. The 18S gene (˜200 bp) is usedas an internal control. The primers used for each sequence, PCR reactionconditions, and the expected amplicon size are contained in FIG. 2.Crossing point value, which is the point at which the fluorescencecrosses the threshold, and melting curve analyses are noted. The meltingcurve data are collected for all genes to ensure a single peak,indicating amplification of a specific region by a pair of primers. Therelative expression values are calculated using the 2(-Delta C(T))method (Livak and Schmittgen, Methods, 25:402-408 (2001)). See Table 3below.

TABLE 3 P. argentatum Average Relative Genotypes Expression of Aos G7-111.02 ± 0.2 pND6-10 1.14 ± 0.2 pND6-12 1.02 ± 0.3 pND6-29 1.04 ± 0.2pND6-32 1.00 ± 0.3 pND6-33 1.11 ± 0.3 pND6-35 0.90 ± 0.2 pND6-41 0.91 ±0.2 pND6-AosiL₅₋₁ 0.39 ± 0.1* pND6-AosiL₇₋₂ 0.49 ± 0.1* pND6-AosiL₈₋₁0.53 ± 0.1* pND6-AosiL₉₋₁₅ 0.44 ± 0.1* pND6-AosiL₉₋₁₆ 0.37 ± 0.04*pND6-AosiL₁₂₋₁ 0.55 ± 0.1* pND6-AosiL₁₂₋₃ 0.48 ± 0.1* pND6-AosiL₁₃₋₂0.55 ± 0.05* pND6-AosiL₁₅₋₃ 0.36 ± 0.1* pND6-AosiL₁₅₋₄ 0.48 ± 0.2*pND6-Aos₄₋₁ 2.15 ± 0.1** pND6-Aos₄₋₂ 2.11 ± 0.3** pND6-Aos₅₋₁ 2.29 ±0.4** pND6-Aos₇₋₁ 2.40 ± 0.6** pND6-Aos₇₋₃ 2.11 ± 0.2** pND6-Aos₈₋₂ 2.15± 0.2** pND6-Aos₁₀₋₁ 2.44 ± 0.7** pND6-Aos₁₀₋₂ 2.12 ± 0.4** pND6-Aos₁₁₋₅2.30 ± 0.3** pND6-Aos₁₄₋₂ 2.23 + 0.1** G7-11 = wild-type control; pND6 =empty vector (pND6 without Aos); pND6-AosiL = PaAos isknocked-down/silenced; pND6-Aos = PaAos is over-expressed Results areaverage of three independent plants, each plant having three technicalreplicates. * and ** indicate significant difference in comparison toG7-11 guayule and/or pND6 (controls) at p > 0.05 and 0.005,respectively.

Next, to gain more insight as to where the PaAos is spatially located,the expression pattern of PaAos in various guayule tissues is analyzedusing qRT-PCR. Total RNA is extracted from leaves, petiole, stem, root,young flower, mature flower, peduncle, stembark of 8-week-oldtissue-cultured genetically altered plants as well as 2-month-oldgreenhouse grown genetically altered plants using the protocol describedabove. qRT-PCR is performed as described above on these samples of totalRNA. Primers (SEQ ID NOs: 7 and 8 in FIG. 2) are designed to amplify˜200 bp PCR product in PaAos coding sequence. The expression level foreach tissue are compared to the tissue cultured and greenhouse leaftissues, respectively. The 18S gene (˜200 bp) (forward primer is SEQ IDNO: 5 and reverse primer is SEQ ID NO: 6, described supra and in FIG. 2)is used as an internal control. As shown in Table 4, infra, the largestlevel of PaAos expression is present in the stem, root and stembarktissues, suggesting that these tissues are sites in which PaAos isfunctioning.

TABLE 4 Growth Conditions: MS Medium Greenhouse Tissue Source RelativeExpression Leaf 0.98 ± 0.1 1.04 ± 0.2 Petiole  0.31 ± 0.06 0.41 ± 0.1Stem 2.27 ± 0.2 3.37 ± 0.4 Root 2.47 ± 0.2 3.74 ± 0.3 Young Flower nodata 1.23 ± 0.4 Mature Flower no data 0.69 ± 0.2 Peduncle no data 0.25 ±0.1 Bark no data 4.49 ± 1.2 The error bars represent tissues collectedfrom 3 individual plants.

Example 3. Rubber Quantification in Tissue

Rooted plantlets (genetically altered, empty vector transformed (pND6without PaAos), and wild-type control) from transferred shoot tips aregrown on half-strength MS medium (PhytoTechnology Laboratories, OverlandPark, Kans.) in Magenta boxes (Caisson Labs, Smithfield, Utah) for 6weeks. The top part of the plantlets are separated from the medium andlyophilized for 48 hours. The dried tissues are placed in a 50 mLstainless steel grinding jar containing grinding ball, frozen in liquidnitrogen for 5 minutes and finely ground using the Retsch mixer mill MM400 at a frequency of 30/second for 1 minute (Verder Scientific Inc.,Newtown, Pa.). Three hundred milligrams (0.3 g) of pulverized tissuesare partitioned with Ottawa sand (Fisher Scientific, Fair Lawn, N.J.)and loaded into 11 mL stainless steel extraction cells (Dionex,Sunnyvale, Calif.). Three sequential extractions are performed using theAccelerated Solvent Extractor (ASE 2000; Dionex, Sunnyvale, Calif.): 1.Acetone: to remove resinous material and the low molecular weightorganic solubles; 2. Methanol: to remove chlorophyll and otheralcohol-soluble materials; 3. Cyclohexane: to remove rubber. Naturalrubber is quantified gravimetrically. The percent (%) rubber is theamount (% dw) of cyclohexane extract from 0.3 g dried tissue. ThepND6-AosiL plants have 1.5 to 2 times more rubber than G7-11, pND6 andpND6-Aos in tissue-cultured environment (Table 5). In Table 5, therubber content is quantified from leaf and stems of the indicatedguayule genotypes grown in MS media.

TABLE 5 Rubber content of guayule plant shoots determined by AcceleratedSolvent Extraction P. argentatum Genotypes Average Rubber Content (%)G7-11.1 1.01 ± .01 G7-11.2 1.11 ± .02 pND6-12 1.13 ± 0.2 pND6-33 1.10 ±0.1 pND6-35 1.04 ± 0.2 pND6-AosiL₅₋₁  1.8 ± 0.1* pND6-AosiL₇₋₂  2.0 ±0.3** pND6-AosiL₈₋₁  2.1 ± 0.04*** pND6-AosiL₈₋₂  1.7 ± 0.1**pND6-AosiL₉₋₁₅  1.7 ± 0.02** pND6-AosiL₉₋₁₆  2.3 ± 0.4* pND6-AosiL₁₂₋₁2.46 ± 0.3* pND6-AosiL₁₂₋₃ 1.62 ± 0.002*** pND6-Aos₄₋₁ 0.96 ± 0.2pND6-Aos₄₋₂ 0.85 ± 0.1 pND6-Aos₅₋₁ 1.09 ± 0.1 pND6-Aos₅₋₂ 1.23 ± 0.1pND6-Aos₇₋₁ 0.96 ± .02 pND6-Aos₈₋₂ 1.23 ± 0.1 pND6-Aos₁₁₋₅ 1.23 ± 0.1Note: The rubber content is quantified from shoots (leaves + stems) ofguayule genotypes grown in MS media. Error bars represent threebiological plants with three technical replicates each. *, ** and ***indicate significant difference in comparison to G7-11 guayule and/orpND6 (controls) at p > 0.05, 0.005 and 0.0005, respectively.

Next, the genetically altered guayule plants are transplanted into soiland grown for 2 months under 27° C. (16 h)/25° C. (8 h) and 27° C. (16h)/10° C. (8 h) in growth chamber conditions, representing a microcosmof what guayule plants experience in the field during winter. Underthese conditions, pND6-AosiL plants also exhibited elevated rubbercontent, having up to 31% times more rubber in comparison with G7-11,pND6 and pND6-Aos plants (Table 6). In Table 6, the rubber content isquantified from shoots and roots of the indicated guayule genotypesgrown in soil. These plants are approximately 4 months old when rubbercontent is analyzed (tissue culture (approx. 1.5 months), greenhouse(approx. 1 month), and growth chamber (approx. 2 months)).

TABLE 6 Rubber content of guayule plant tissue determined by AcceleratedSolvent Extraction Average Rubber Content (%) Shoot Root P. argentatum27° C. (16 h)/ 27° C. (16 h)/ 27° C. (16 h)/ 27° C. (16 h)/ Genotypes25° C. (8 h) 10° C. (8 h) 25° C. (8 h) 10° C. (8 h) G7-11.1 1.22 + 0.091.13 + 0.11 1.10 + 0.04 0.95 + 0.10 G7-11.2 1.06 + 0.12 1.37 + 0.090.63 + 0.12 0.71 + 0.09 pND6-12 1.04 + 0.18 1.31 + 0.06 0.65 + 0.070.72 + 0.16 pND6-33 0.90 + 0.06 1.14 + 0.12 0.62 + 0.08 0.82 + 0.10pND6-35 1.18 + 0.08 1.27 + 0.10 1.09 + 0.06 0.94 + 0.03 pND6-AosiL₇₋₂1.49 + 0.07*** 1.86 + 0.11*** 0.56 + 0.03 1.26 + 0.09** pND6-AosiL₈₋₁1.48 + 0.05*** 1.91 + 0.07*** 0.78 + 0.14 1.19 + 0.04** pND6-AosiL₉₋₁₆1.46 + 0.04*** 2.01 + 0.08*** 0.66 + 0.12 1.16 + 0.02** pND6-AosiL₁₂₋₁1.55 + 0.07* 1.80 + 0.05** 1.26 + 0.04*** 1.52 + 0.07** pND6-Aos₄₋₁0.97 + 0.26 1.13 + 0.2 0.57 + 0.05 0.79 + 0.09 pND6-Aos₄₋₂ 1.21 + 0.101.30 + 0.08 1.05 + 0.10 0.94 + 0.11 pND6-Aos₅₋₁ 0.98 + 0.30 1.04 + 0.120.54 + 0.11 0.62 + 0.10 pND6-Aos₇₋₁ 0.96 + 0.30 1.15 + 0.14 0.57 + 0.060.55 + 0.08 Note: The rubber content is quantified from shoots and rootsof guayule genotypes grown in soil. Plants are transferred to soil fromtissue culture and are grown in a growth chamber environment. Error barsrepresent three biological plants with three technical replicates each.*, ** and *** indicate significant difference in comparison to G7-11and/or pND6 (controls) at p > 0.05, 0.005 and 0.0005, respectively.

Because rubber is also accumulated in root tissue, the rubber content inthe root tissues is also quantified. For the root rubber content, theconsistent, significant difference is only under 27° C. (16 h)/10° C. (8h) in which pND6-AosiL guayule have an increased in rubber contentcompared with the controls and pND6-Aos (Table 6). The data in Table 6demonstrate that the combination of cold temperature and silencing PaAosis synergistic, causing guayule to produce a greater amount of rubberthan guayule exposed to just cold temperature or to just PaAossilencing. For example, cold treatment alone increased shoot rubbercontent in the control (pND6-12) by 19%—from an average of 1.04% to1.24%. But cold treatment of the Aos-downregulated plants (pND6-AosiL)increased rubber by 27%—from average 1.50% to 1.90%. In root tissues,cold treatment increased the rubber content for the control (pND6-12) by5.1% (from 0.79 to 0.83% rubber), but cold treatment of theAos-downregulated plants (pND6-AosiL) increased rubber by 57%—fromaverage 0.82% to 1.28%. See Table 7, infra. From the ASE results, theincreased in rubber content is very apparent in the pND6-AosiLgenotypes.

TABLE 7 Average Rubber Content (%) Shoot Root 27° C. 27° C. 27° C. 27°C. P. argentatum (16 h)/ (16 h)/ (16 h)/ (16 h)/ Genotypes 25° C. (8 h)10° C. (8 h) 25° C. (8 h) 10° C. (8 h) pND6-12 1.04 1.24 0.79 0.83pND6-AosiL₇₋₂ 1.50 1.90 0.82 1.28 pND6-Aos₄₋₁ 1.03 1.16 0.68 0.73

Example 4. Protein Detection in Rubber Particles

Guayule washed rubber particles (WRPs) are isolated from geneticallyaltered guayule lines (pND6-AosiL and pND6-Aos) and non-altered guayuleusing the protocol set forth in Cornish and Backhaus, Phytochemistry,29: 3809-3813 (1990). Rubber particles are extracted from non-alteredand genetically altered 1 year old greenhouse plants. First, ˜60 g to˜70 g of stembark tissues are peeled off from the plant, grounded with ablender containing cold-extraction buffer, and further purified withcold-washed buffer three times by centrifugation. The protein extracts(1 mg) are run on an SDS-PAGE and detected with silver staining. On theSDS-PAGE gel, endogenous Aos protein runs as ˜53 kDa in the non-alteredand overexpressed plants but not in the RNAi lines. To determine the dryweight of the WRPs, 50 μL of the protein extracts are aliquoted 3× on aweighing paper, oven-dried over-night in a 60° C. incubator and weighedthe next day. Generally, approximately 0.5 mg/μL to approximately 1.5mg/μL WRPs are extracted.

Example 5. Hormone Production

PaAos is an enzyme in the biosynthetic pathway that produces severaldifferent plant hormones, including jasmonic acid, SA, abscisic acid,gibberellin A₂₀, gibberellin A₁, and gibberellin A₃. As such, the amountof these hormones is quantified in genetically altered (pND6-AosiL andpND6-Aos), empty vector transformed (pND6 without PaAos; control), andwild-type (G7-11, control) tissue-cultured guayule plants using theprotocol described in Pan et al., Nature Protocols 5:986-992 (2010). SeeTable 8, infra. Briefly, leaves and stems are snap-frozen and ground topowder with mortar and pestle. Solvent extraction solution containing2-propanol/H₂O/concentrated HCl (2:1:0.002; vol/vol/vol) and internalstandards are added to ˜50 mg of pre-weighed tissues. After solventextraction, sample concentration and re-dissolution, 50 μL of the samplesolution is placed into the liquid chromatography-tandem spectrometry(Agilent GC-MS 5977A; Agilent Technologies, Santa Clara, Calif.) forhormone analysis. Three biological plants, with three technicalreplicates of each plant, are used.

TABLE 8 Concentration (ng/gfw) P. argentatum Jasmonic Salicylic AbscisicGibberellin Gibberellin Gibberellin Genotypes Acid Acid Acid A₂₀ A₁ A₃G7-11 5.36 ± 1.2 5.50 ± 0.8  11.01 ± 1.9  15.95 ± 0.7  9.95 ± 0.07 3.52± 0.2  pND6-12 1.57 ± 0.1 4.89 ± 0.6  7.05 ± 0.8 14.11 ± 1.2 12.39 ± 2.22.19 ± 0.01 pND6-33 4.76 ± 1.0 5.04 ± 0.1  7.24 ± 0.3 13.86 ± 2.6 12.70± 2.3 1.79 ± 0.09 pND6-35 1.96 ± 0.4 5.6 ± 0.5 7.10 ± 0.6 14.41 ± 1.214.65 ± 0.2 2.16 ± 0.3  pND6-AosiL₇₋₂  0.57 ± 0.1** 9.51 ± 0.5*  4.71 ±0.6*  9.63 ± 1.2*  5.13 ± 0.8* 0.86 ± 0.3* pND6-AosiL₉₋₁₆   0.57 ±0.01** 7.65 ± 0.2*  2.96 ± 0.3*  8.85 ± 2.1*   7.81 ± 0.2** 0.80 ± 0.3*pND6-AosiL₁₂₋₁   0.68 ± 0.05** 9.65 ± 1.1*  3.64 ± 0.5*  10.59 ± 0.2* 6.75 ± 1.1*  1.32 ± 0.07* pND6-Aos₄₋₁ 1.48 ± 0.2 4.03 ± 0.7   9.13 ±1.71 15.49 ± 0.6 10.78 ± 0.1 1.93 ± 0.06 pND6-Aos₄₋₂ 3.25 ± 0.2 4.76 ±0.6  13.9 ± 1.3 no data 14.63 ± 2.7 1.80 ± 0.1  pND6-Aos₇₋₁ 1.41 ± 0.35.50 ± 0.02 8.84 ± 0.3 13.64 ± 1.2  13.96 ± 0.01 1.84 ± 0.1  ± representthree biological plants with three technical replicates each plant. *and ** indicate significant difference in comparison to G7-11 and/orpND6 (controls) at p > 0.05 and 0.005, respectively.

As evident in Table 8, the amount of jasmonic acid, abscisic acid andgibberellic acids are reduced in the pND6-AosiL guayule compared to theamount in the controls (wild-type (G7-11) and empty vector transformedplants) and pND6-Aos guayule. Conversely, the SA content is elevated inpND6-AosiL compared to the controls and pND6-Aos lines. These resultssuggest that knocking down PaAos expression not only reduces productionof jasmonic acid but also affects the level of other hormones as well.

Example 6. Plant Architecture and Photosynthetic Rates

Three independent events from each of the overexpression (pND6-Aos) andof the silenced (pND6-AosiL) lines; as well as two pND6 and onewild-type (G7-11) controls are selected for further studies. pND6-AosiLplants grown in greenhouse (data not shown) and growth chamberconditions are bigger (see FIG. 3), have darker green leaves (data notshown), and increased chlorophyll measurement than the wild type andother genetically altered plants (see FIG. 4). As demonstrated in FIG.3, under 27° C. (16 h)/25° C. (8 h) and 27° C. (16 h)/10° C. (8 h)environments, pND6-AosiL plants are significantly taller and wider inboth conditions. These plant architectural traits reflect the fact thatpND6-AosiL plants are larger and have more shoot and root biomass.

pND6-AosiL genotypes have also a greater number of stems than thewild-type and empty vector controls. Well-branched guayule plants are anindicator of having increased rubber yield because of the presence ofmore sink tissue available to store rubber. Additionally, the maturestembark tissues in pND6-AosiL have thicker diameter (ranging fromapproximately 35% to approximately 54%) under both 27° C. (16 h)/25° C.(8 h) and 27° C. (16 h)/10° C. (8 h) in comparison to the controls andpND6-Aos. See FIG. 5.

Based on this observation, the photosynthetic rate of the plants ismeasured using LI-COR 6400xt (LI-COR Biosciences, Lincoln, Nebr.) tomeasure the photosynthetic rate. Measurements are taken between 0900 to1200 h. Fully expanded middle leaf are clamped on the Li-Cor head. Afterthe measured and set parameters are stabilized, the reading is taken.The middle leaf position is chosen because this position showssignificant differences based on chlorophyll meter measurements, FIG. 4.(SPAD-502; Minolta Camera Ltd., Japan). The pND6-AosiL plants exhibithigher photosynthetic rate (23-31%) in comparison to G7-11, pND6 andpND6-Aos plants (Table 9, infra). Additional physiological measurementsreveal that pND6-AosiL stomatal limitation is one of the factorsinvolved in the higher photosynthetic rate as pND6-AosiL plants showhigher stomatal conductance and transpiration rate when compared toG7-11, pND6 and pND6-Aos plants (Table 9, infra). Furthermore,chlorophyll fluorescence measurements clearly show PSII and ETRparameters used for elucidating the efficiency of PSII are significantlyhigher than G7-11, pND6 and pND6-Aos plants (Table 9, infra) in both 27°C. (16 h)/25° C. (8 h) and 27° C. (16 h)/10° C. (8 h) treatmentconditions. Having higher PSII and ETR indicate that amount of lightenergy absorbed and carbon assimilated is available more in pND6-AosiLplants to convert into energy for the plant to use (i.e., growth anddevelopment as well as rubber production) compared to the controls andpND6-Aos plants. Meanwhile, the NPQ measurements for the pND6-AosiLplants are lower under the 27° C. (16 h)/25° C. (8 h) condition andhigher under the 27° C. (16 h)/10° C. (8 h) treatment in comparison withthe controls and pND6-Aos plants. Having higher NPQ suggests thatpND6-AosiL have improved heat dissipation ability compared to G7-11,pND6 and pND6-Aos plants which could help prevent lipid or other cellmembrane damage under environmental stress.

TABLE 9 P. argentatum Genotypes Pn g € ΦPSII ETR NPQ 27° C. (16 h)/25°C. (8 h) G7-11 5.75 ± 0.8 0.093 ± 0.03 2.33 ± 0.6 0.146 ± 0.015 115.73 ±11.3  1.97 ± 0.2 pND6-10 6.29 ± 0.7 0.110 ± 0.03 2.74 ± 0.6 0.141 ±0.015 111.1 ± 11.6 1.74 ± 0.1 pND6-12 6.20 ± 0.8 0.109 ± 0.02 2.66 ± 0.50.145 ± 0.015 114.4 ± 12.1 1.90 ± 0.2 pND6-AosiL₇₋₁   8.56 ± 0.6***  0.147 ± 0.01***   3.41 ± 0.3***  0.196 ± 0.027*   165.2 ± 10.7***  1.33 ± 0.2*** pND6-AosiL₈₋₁   8.40 ± 0.6***   0.155 ± 0.02***   3.67 ±0.4***  0.180 ± 0.008*  141.4 ± 6.2**   1.27 ± 0.2*** pND6-AosiL₉₋₁₆  7.96 ± 0.5***   0.162 ± 0.03***   3.57 ± 0.5***  0.186 ± 0.018*  134.4± 3.3**   1.19 ± 0.3*** pND6-Aos₄₋₁ 5.62 ± 0.9 0.110 ± 0.04 2.59 ± 0.70.133 ± 0.008 104.7 ± 6.7  1.92 ± 0.3 pND6-Aos₅₋₁ 5.70 ± 0.8 0.110 ±0.04 2.60 ± 0.7 0.133 ± 0.008 104.7 ± 6.7  1.95 ± 0.3 pND6-Aos₇₋₁ 5.87 ±0.9 0.113 ± 0.04 2.65 ± 0.7 0.137 ± 0.008 107.7 ± 6.3  2.07 ± 0.4 27° C.(16 h)/10° C. (8 h) G7-11 2.23 ± 0.5 0.054 ± 0.02 1.31 ± 0.4 0.070 ±0.007 55.4 ± 5.4 1.58 ± 0.2 pND6-10 2.04 ± 0.4 0.057 ± 0.02 1.55 ± 0.30.064 ± 0.006 50.7 ± 4.9 1.56 ± 0.2 pND6-12 2.28 ± 0.4 0.065 ± 0.03 1.67± 0.7 0.066 ± 0.003 51.6 ± 2.7 1.51 ± 0.3 pND6-AosiL₇₋₁   4.14 ± 0.4***  0.104 ± 0.02***   2.54 ± 0.4***   0.104 ± 0.011***   81.0 ± 8.2*** 2.29 ± 0.3** pND6-AosiL₈₋₁   4.15 ± 0.4***   0.112 ± 0.05***   2.71 ±0.8***   0.102 ± 0.013***   77.0 ± 4.5***  2.33 ± 0.2** pND6-AosiL₉₋₁₆  4.14 ± 0.6***   0.101 ± 0.03***   2.60 ± 0.5***   0.101 ± 0.009***  79.4 ± 6.9***  1.95 ± 0.1** pND6-Aos₄₋₁ 2.27 ± 0.5 0.059 ± 0.02 1.51 ±0.3 0.065 ± 0.011 60.4 ± 5.5 1.35 ± 0.3 pND6-Aos₅₋₁ 2.97 ± 0.3 0.069 ±0.01 1.81 ± 0.2 0.077 ± 0.007 58.5 ± 6.3 1.25 ± 0.2 pND6-Aos₇₋₁ 2.45 ±0.6 0.063 ± 0.01 1.93 ± 0.3 0.065 ± 0.010 50.1 ± 5.8 1.44 ± 0.2 Pn = netphotosynthetic rate; g = stomatal conductance; € = Transpiration rate;ΦPSII = Efficiency of Photosystem II; ETR = Electron Transport Rate; NPQ= Non-photochemical quenching *, ** and ***indicate significantdifference in comparison to G7-11 and/or pND6 (controls) at p > 0.05,0.005 and 0.0005, respectively.

Example 7. Quality of Natural Rubber from pND6-AOSiL Plants

The length of the polymer chain, a.k.a. rubber molecular weight, is theprimary determinant of quality in natural rubber. Gel permeationchromatography (GPC) is used to measure the molecular weight of rubberfrom guayule tissue culture plants' extracts. Cyclohexane extractablescollected from ASE (see Example 3 and Table 5 supra) are re-suspended inapproximately 3 mL of tetrahydrofuran (THF) overnight with gentleshaking (Multi-Purpose Rotator. Thermo Scientific, Waltham. Mass.). Thesolution is syringe-filtered through a 1.6 μm glass microfiber GF/Afilter (Whatman GE Healthcare, Piscataway, N.J.), then injected into aHewlett Packard 1100 series HPLC (1.0 mL/min flow rate, 50 μL injectionvolume, THF continuous phase) and size exclusion separated by twoAgilent PL gel 10 μm Mixed-B columns in series (35° C.) (Santa Clara,Calif.). The resulting chromatograms are used to calculate the rubbermolecular weight shown in FIG. 6 (using Astra software (Wyatt TechnologyCorp., Santa Barbara, Calif.)). The molecular weight of natural rubberfrom three pND6-AosiL transformed guayule plants (silenced) is greaterthan from wild-type guayule line G7-11, two negative control pND6transformed guayule plants and three pND6-Aos transformed guayule plants(overexpressed) indicating better quality rubber in the PaAos silencedguayule plants. In FIG. 6, the asterisks (*) and (**) above the threepND6-AosiL transformed guayule plant lines indicate significantdifference in comparison to the negative control pND6 transformedguayule plant lines at p>0.05 and p>0.005, respectively.

Example 8. PaAos SNPs Change Protein's Functionality

Eight different guayule cultivars grown in tissue culture are evaluatedfor their rubber content and expression of PaAos gene. The combinationof ASE method (see Example 3 supra) and qRT-PCR (see the protocols andprimers discussed in Example 2, supra, and FIG. 2) are used to comparerubber content to the level of PaAos gene expression. First, seeds fromguayule lines (PI 478648, W6 549, PI 478651, PI 478652, PI 478653, PI478654, PI 478655, and PI 478662) are obtained from a public germplasmbank (USDA-ARS National Plant Germplasm System, Parlier, Calif.). Theseeds are germinated and plants grown in tissue culture medium for 8weeks. The natural rubber content is determined by standard methods, asdescribed previously (ASE). The rubber content varied significantlybetween lines, from 0.95% to 1.73% (Table 10). Cultivar (line) W6 549has the lowest average rubber content (%) and cultivar PI 478652 has thelargest average rubber content (%) (see Table 10).

TABLE 10 P. argentatum Germplasm Genotypes name Average Rubber Content(%) PI 478648 11635 1.38 ± 0.08 W6 549 CAL 7 0.95 ± 0.03 PI 478651 117011.40 ± 0.13 PI 478652 12229 1.73 ± 0.28 PI 478653 12231 1.36 ± 0.11 PI478654 N396 1.20 ± 0.05 PI 478655 N565 0.98 ± 0.01 PI 478662 A48118 1.07± 0.06

PaAos gene expression for cultivars W6 549 and PI 478652 are determinedby standard methods (qRT-PCR, see Example 2, supra). Shoot tissues (leafand stem) are collected into 2 mL tubes and are snap-frozen in liquidnitrogen and then hand pulverized (mortar and pestle). RNA is extractedusing the TRIzol® method (Ambion, Pittsburgh, Pa.) using manufacturer'srecommended protocol. The RNA concentration is quantified with theNanoDrop ND1000 (ThermoScientific, Wilmington, Del.). An RNA cleanup isperformed using RNeasy MinElute Cleanup kit using manufacturer'srecommended protocol (Qiagen Inc., Valencia, Calif.). The RNA is elutedwith 30-50 tit of RNase-free water along with on-column DNase1treatment.

An iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif.) is used tosynthesize complementary DNA (cDNA) from the isolated RNA. An amount of1 μg of RNA is used in the 20 mL reaction mixture. For semi-qRT-PCR, 2μL of the diluted cDNA (1:20) is used in a 50 μL reaction mixture of OneTaq Quick-Load 2× Master Mix with Standard Buffer (New England Biolabs,Inc., Ipswich, Mass.) with the following forward and reverse primers (10μM) 5′-ATGGACCCATCGTCTAAACCC-3′ (SEQ ID NO: 16) and5′-TCATATACTAGCTCTCTTCAGG-3′ (SEQ ID NO: 17), respectively. Thesemi-qRT-PCR is run using Eppendorf thermocyler (ThermoFisherScientific, Waltham, Mass.) with the following thermal cycle: 94° C.pre-incubation for 30 seconds; amplification for 35 cycles at 94° C. for30 seconds, annealing at 58° C. for 1.5 minutes, and extension at 68° C.for 1 minute. The final extension time is for 5 minutes at 68° C. Eachsemi-qRT-PCR run is performed with three independent tissue samples. The18S gene (˜200 bp) is used as an internal control using SEQ ID NO: 5 asforward primer and SEQ ID NO: 6 as reverse primer. PCR products (˜1.4Kbp) are separated by electrophoresis on a 1% (w/v) agarose gel. Resultsclearly demonstrate that W6 549 (“W6549”) cultivar has increased PaAosexpression compared to PI 478652 (“478652”) cultivar (see FIG. 7). Theseresults suggest that the rubber content is inversely correlated with thePaAos gene expression.

PaAos coding sequence in the two lines, W6 549 cultivar (low rubberproducer) and PI 478652 cultivar (high rubber producer) are determinedby PCR, and the sequences are compared to G7-11 cultivar sequence.Extraction of the cDNA from the agarose gel is performed with QIAquickGel Extraction kit (Qiagen, Germantown, Md.) using manufacturer'srecommended protocol. The 1.4 kb band visualized with ethidium bromideis excised from the gel with a clean razor blade. After determining theweight of the gel slice, 300 μl of Buffer QG pH 7.5 is added for every100 mg of gel slice with the DNA fragment size of 100 bp-4 kb. To bindthe DNA, 30 μl QIAEX II beads are added per 5 μg of DNA. The resuspendedgel is dissolved by incubation at 50° C. for 10 minutes with vigorousvortexing every 2 minutes. Each sample is centrifuged at 16,110×g in aconventional table top microcentrifuge for 30 seconds. Aftercentrifugation, each sample rests at room temperature for five minutes.The supernatant is discarded, and the pellet is washed with cold 750 μlBuffer PE twice. The pellet is air dried until it turned solid white.The DNA is resuspended with 50 μl of 10 mM of Tris-Cl pH 8.5. Thedissolved DNA pellet stands at room temperature for 1 minute prior tocentrifugation. The supernatant is collected as the purified cDNAproduct. To confirm the integrity of the sequence, three independent PCRproducts are sent to Elim Biopharmaceuticals (Hayward, Calif.) foranalysis. The sequence alignment is performed using softwares MEGA 6.06(Tamura, et al., Mol. Bio. and Evol., 30:2725-2729 (2013)) and T-Coffee(Notredame, et al., J. Mol. Biol., 302:205-217 (2000)). As evident inFIGS. 8A-8C, a few SNPs exist which give rise to changes in the aminoacid sequences (see FIG. 9). In particular, the amino acids at positions318, 408 and 459 are D, I and L in W6 549 (“W6549”) cultivar while PI478652 (“478652”) cultivar has N, V and W, respectively (FIG. 9). Thesedifferences in three amino acids result in different PaAos functionalitywhich result in different amounts of rubber being produced.

By reducing PaAos' functionality, one increases rubber production inguayule. As discussed previously, reducing the amount of PaAos bysilencing PaAos expression or translation increases rubber production.Null mutations (such as, but not limited to, insertions that disrupttranslation of a functional protein, changing slice site recognitionnucleotide(s), and changing ATG initiation codon) alter the productionof functional PaAos which result in an increase in rubber production.Alternations in PaAos' DNA sequence that result in specific amino acidchanges within PaAos also increase rubber production. For example, DNAalterations that change PaAos sequence from D318, I408 and/or L459(present in W6 549 cultivar, low rubber producer) to N318, V408 and W459(present in PI 478652 cultivar, high rubber producer) (or any othernon-conservative amino acid for D318, I408, and/or L459) result in anincrease in rubber production because of a decrease in PaAosfunctionality. In addition, altering PaAos' DNA sequence encoding S332,E336, R339, S359, and/or S411 to a sequence encoding non-conservativeamino acids results in reducing PaAos' functionality and thus increasingrubber production. See, Pan, et al., J. of Bio. Chem.,273(29):18139-18145 (1998), contents of which are expressly incorporatedby reference.

Based on these results from these assays, one can screen for thepresence of particular SNPs in the PaAos gene in various guayulevarieties at the seedling stage to determine if a particular guayulevariety is a high rubber producer or low rubber producer. To screenguayule, one obtains a tissue sample from the guayule to be screened,isolates the sample's mRNA or total RNA, and conduct a PCR assay(regular PCR or RT-PCR or qRT-PCR) using PaAos primers that surround thenucleotides encoding amino acids N318, V408 and W459 to identify guayuleplants encoding these amino acids which indicate that the guayuleproduces more rubber than a guayule not having these amino acids withinPaAos. Guayule seedlings (plants that are between 2-4 weeks and 8-10weeks post-germination) are screened. Alternatively, guayule plants thatare approximately 2 or 3 months old can be screened. While any planttissue can be used to conduct the SNP analysis, bark and leaves may beeasier to sample than other tissue (such as roots). Using the two primerpairs 5′-CCTACTCGACGCCAAGAG-3′ (forward, SEQ ID NO: 18) and5′-TTCAGCTGAGCATGTCTAGGT-3′ (reverse, SEQ ID NO: 19) and5′-GGCATTGTTGAAGTACATATGG-3′ (forward, SEQ ID NO: 20) and5′-CCAAAGGAGACTCGCCTAATT-3′ (SEQ ID NO: 21), one determines if theseedling or plant contains D318, I408 and/or L459 (similar to G7-11 andW6 549 cultivars) and thus produces less rubber than a “high rubberproducer” plant. Alternatively, using the two primer pairs5′-CCTACTCGACGCCAAAAGC-3′ (forward, SEQ ID NO: 22) and5′-CTTAAGTTGAGCATGTCTAGGTT-3′ (reverse, SEQ ID NO: 23) and5′-GGCATTGTTGAAGTACGTATGG-3′ (forward, SEQ ID NO: 24) and5′-CCCAAGGAGACTCGCCTA-3′ (reverse, SEQ ID NO: 25), one determines if theseedling or plant contains N318, V408 and/or W459 (similar to PI 478652cultivar) and thus produces more rubber than a “low rubber producer”plant. Furthermore, guayule containing PaAos with S332, E336, R339,S359, and/or S411, in combination with one or more of D318, I408 andL459, are also low rubber producing plants. Primers are designed tocover the SNPs for these amino acids which are used to identify lowrubber producing guayule. Similarly, guayule containing PaAos withnon-conservative amino acids to D318, S332, E336, R339, S359, I408,S411, or L459, or a combination thereof, are high rubber producingplants, and primers are designed to cover the SNPs for these amino acidswhich are used to identify high rubber producing plants. Afterperforming the PCR assay, one isolates the amplicon(s) and sequences theamplicon(s) to determine the presence or absence of the indicated SNPs.Other techniques are known to one of ordinary skill in the art foridentifying amplicons with the indicated SNPs.

Alternatively, an ELISA or other type of antibody assay can distinguishbetween PaAos containing N318, V408 and/or W459 (PI 478652 cultivar(high producer)) and PaAos containing D318, I408 and/or L459 (W6 549cultivar (low producer)), with or without one or more of S332, E336,R339, S359, and S411. An ELISA using a monoclonal antibody (mAb) that isspecific for PaAos containing N318, V408 and/or W459, with or withoutone or more non-conservative amino acids substituted for S332, E336,R339, S359, and S411, would identify high rubber producing plants.Alternatively, an ELISA using a mAb that is specific for PaAoscontaining D318, I408 and/or L459 with or without one or more aminoacids S332, E336, R339, S359, and S411, would identify low rubberproducing plants. Protein isolated from tissue sample, as describedabove, can be contacted with the mAb(s) in the ELISA which then changescolor to indicate the presence of PaAos having the particular aminoacids and structure to which the mAb binds.

Guayule encoding PaAos with conservative amino acid substitutions forN318, V408 and/or W459 (and optionally with non-conservative amino acidsubstitutions for S332, E336, R339, S359, and/or S411) are high rubberproducing guayule. Similarly, guayule encoding PaAos with conservativeamino acid substitutions for D318, I408 and/or L459 (and optionally withor without conservative amino acid substitutions for S332, E336, R339,S359, and/or S411) are low rubber producing guayule. See Table 2 andpreceding paragraph for information about conservative andnon-conservative amino acid substitutions, and Table 1 for DNA codonsfor amino acids.

One can generate DNA mutations within guayule seed's genome using EMS,UV light, protons, or other known mutagens to create altered guayuleseeds. Then one germinates the seeds into seedlings and screen theseedlings for PaAos mutations which reduce PaAos' functionality. In oneembodiment, the above described primers are used to determine if theindicated SNPs are present in the altered guayule seedling so that onedoes not need to grow the altered guayule for years before determiningif the altered guayule is likely a high rubber producer or a low rubberproducer. One can screen for D318, S332, E336, R339, S359, I408, S411,and/or L459 and conservative amino acids to determine if the alteredseedling is a low rubber producer; or screen for non-conservative aminoacid substitutions to determine if the altered seedling is a high rubberproducer.

The foregoing detailed description and certain representativeembodiments and details of the invention have been presented forpurposes of illustration and description of the invention. It is notintended to be exhaustive or to limit the invention to the precise formsdisclosed. It will be apparent to practitioners skilled in the art thatmodifications and variations may be made therein without departing fromthe scope of the invention. All references cited herein are incorporatedby reference.

We, the inventors, claim as follows:
 1. An altered guayule, parts andprogeny thereof, that produces more rubber than amount of rubberproduced by a non-altered guayule comprising a mutation in PaAos;wherein said mutation is selected from the group consisting of (i) analteration in a PaAos codon encoding an amino acid to a stop codon, (ii)an alteration of PaAos' translation initiation codon to another codon,(iii) an alteration in PaAos ribosome binding site's sequence, (iv) analteration in one or more PaAos splice site codons, (v) a deletion ofpart or all of said PaAos' sequence, (vi) an insertion of DNA intoPaAos, and (vii) an alteration in one or more PaAos DNA codon sequencesto encode a non-conservative amino acid; wherein said mutation reducessaid altered PaAos' functionality compared to amount of PaAosfunctionality in said non-altered guayule; wherein said reduced PaAosfunctionality causes said altered guayule to produce an increased amountof rubber compared to said amount of rubber produced by said non-alteredguayule.
 2. The altered guayule of claim 1; wherein said alteration inone or more PaAos DNA codon sequences to encode a non-conservative aminoacid occurs at amino acids located at 318, 332, 336, 339, 359, 408, 411,and 459 with said PaAos sequence.
 3. The altered guayule of claim 2;wherein said non-conservative amino acid substitution excludes at leastone of N318, V408 and W459.
 4. An altered cell of said altered guayuleof claim 1, wherein said altered cell comprises said mutation in PaAos.5. An altered germplasm of said altered guayule of claim 1, wherein saidaltered germplasm comprises said mutation in PaAos.
 6. An altered seedof said altered guayule of claim 1, wherein said altered seed comprisessaid mutation in PaAos.
 7. A method for producing an altered guayulethat comprises a mutated PaAos and produces an increased amount ofrubber compared to amount of rubber produced by a non-altered guayule,said method comprising exposing a non-altered guayule cell or seed to amutagen to produce a mutated guayule cell or seed with said mutatedPaAos; selecting said mutated guayule cell or seed comprising saidmutated PaAos, wherein said mutated PaAos encodes an altered PaAoshaving reduced functionality compared to a non-altered PaAos'functionality; and growing said selected mutated guayule cell or seedcomprising said mutated PaAos to produce an altered guayule thatproduces said altered PaAos with reduced functionality and saidincreased amount of rubber compared to said amount of rubber produced bysaid non-altered guayule.
 8. The method of claim 7, wherein said mutatedPaAos comprises at least one of (i) an alteration in a PaAos codonencoding an amino acid to a stop codon, (ii) an alteration of PaAos'translation initiation codon to another codon, (iii) an alteration inPaAos ribosome binding site's sequence, (iv) an alteration in one ormore PaAos splice site codons, (v) a deletion of part or all of saidPaAos' sequence, (vi) an insertion of DNA into PaAos, and (vii) analteration in one or more PaAos DNA codon sequences to encode anon-conservative amino acid.
 9. The method of claim 8, wherein saidalteration in one or more PaAos DNA codon sequences to encode anon-conservative amino acid occurs at amino acids located at 318, 332,336, 339, 359, 408, 411, and 459 with said PaAos sequence.
 10. Analtered cell of said altered guayule produced according to said methodof claim
 7. 11. An altered germplasm of said altered guayule producedaccording to said method of claim
 7. 12. An altered seed of said alteredguayule produced according to said method of claim 7, wherein saidaltered seed comprises said mutated PaAos.
 13. A method of producing apopulation of high rubber producing guayule plants or seeds comprisingPaAos with low functionality, said method comprising: genotyping a firstpopulation of guayule plants or seeds, said first population of saidguayule plants or seeds comprising said PaAos with low functionality;selecting from said first population one or more guayule plants or seedscomprising said PaAos with low functionality based said genotyping; andproducing from said selected one or more guayule plants or seedscomprising said PaAos with low functionality a second population ofguayule plants or seeds comprising said PaAos with low functionality.14. The method of claim 13, wherein said PaAos with low functionalitycomprises at least one amino acid selected from group of N318, V408,W459, conservative amino acids substitutions thereof, andnon-conservative amino acid substitutions at S332, E336, R339, S359, andS411.
 15. A method of identifying a high rubber producing guayulecomprising detecting PaAos with low functionality in a test guayule,wherein when said test guayule contains PaAos with amino acids N318,V408 and W459, then said test guayule is a high rubber producingguayule.
 16. The method of claim 15, wherein said detecting stepcomprises contacting said PaAos from said test guayule with monoclonalantibodies that bind to PaAos with amino acids N318, V408 and W459; anddetermining if said monoclonal antibodies binds to said test guayulePaAos.
 17. The method of claim 15, wherein said detecting step comprisesobtaining nucleic acids from said test guayule; performing a PCR assaywith said obtained nucleic acids, primer sets having SEQ ID NOs: 22 and23 and SEQ ID NOs: 24 and 25, and a label; determining if an amplicon isgenerated, wherein when said amplicon is produced, then said testguayule contains a PaAos that encodes a PaAos having amino acids N318,V408 and W459, and said test guayule is a high rubber producing guayule.